Contrast agents

ABSTRACT

Ultrasonic visualisation of a suject, particularly of perfusion in the myocardium and other tissues, is performed using novel gas-containing contrast agent preparations which promote controllable and temporary growth of the gas phase in vivo following administration and can therefore act as deposited perfusion tracers. The preparations include a coadministerable composition comprising a diffusible component capable of inward diffusion into the dispersed gas phase to promote temporary growth thereof. In cardiac perfusion imaging the preparations may advantageously be coadministered with vasodilator drugs such as adenosine in order to enhance the differences in return signal intensity from normal and hypoperfused myocardial tissue respectively.

This invention relates to ultrasound imaging, more particularly to novelcontrast agent preparations and their use in ultrasound imaging, forexample in visualising tissue perfusion.

It is well known that contrast agents comprising dispersions ofmicrobubbles of gases are particularly efficient backscatterers ofultrasound by virtue of the low density and ease of compressibility ofthe microbubbles. Such microbubble dispersions, if appropriatelystabilised, may permit highly effective ultrasound visualisation of, forexample, the vascular system and tissue microvasculature, often atadvantageously low doses.

The use of ultrasonography to measure blood perfusion (i.e. blood flowper unit of tissue mass) is of potential value in, for example, tumourdetection, tumour tissue typically having different vascularity fromhealthy tissue, and studies of the myocardium, e.g. to detect myocardialinfarctions. A problem with the application of existing ultrasoundcontrast agents to cardiac perfusion studies is that the informationcontent of images obtained is degraded by attenuation caused by contrastagent present in the ventricles of the heart.

The present invention is based on the finding that ultrasonicvisualisation of a subject, in particular of perfusion in the myocardiumand other tissues, may be achieved and/or enhanced by means ofgas-containing contrast agent preparations which promote controllableand temporary growth of the gas phase in vivo following administration.Thus, for example, such contrast agent preparations may be used topromote controllable and temporary retention of the gas phase, forexample in the form of microbubbles, in tissue microvasculature, therebyenhancing the concentration of gas in such tissue and accordinglyenhancing its echogenicity, e.g. relative to the blood pool.

It will be appreciated that such use of gas as a deposited perfusiontracer differs markedly from existing proposals regarding intravenouslyadministrable microbubble ultrasound contrast agents. Thus it isgenerally thought necessary to avoid microbubble growth since, ifuncontrolled, this may lead to potentially hazardous tissueembolisation. Accordingly it may be necessary to limit the doseadministered and/or to use gas mixtures with compositions selected so asto minimise bubble growth in vivo by inhibiting inward diffusion ofblood gases into the microbubbles (see e.g. WO-A-9503835 andWO-A-9516467).

In accordance with the present invention, on the other hand, acomposition comprising a dispersed gas phase is coadministered with acomposition comprising at least one substance which has or is capable ofgenerating a gas or vapour pressure in vivo sufficient to promotecontrollable growth of the said dispersed gas phase through inwarddiffusion thereto of molecules of gas or vapour derived from saidsubstance, which for brevity is hereinafter referred to as a “diffusiblecomponent”, although it will be appreciated that transport mechanismsother than diffusion may additionally or alternatively be involved inoperation of the invention, as discussed in greater detail hereinafter.

This coadministration of a dispersed gas phase-containing compositionand a composition comprising a diffusible component having anappropriate degree of volatility may be contrasted with previousproposals regarding administration of volatile substances alone, e.g. inthe form of phase shift colloids as described in WO-A-9416739. Thus thecontrast agent preparations of the present invention permit control offactors such as the probability and/or rate of growth of the dispersedgas by selection of appropriate constituents of the coadministeredcompositions, as described in greater detail hereinafter, whereasadministration of the aforementioned phase shift colloids alone may leadto generation of microbubbles which grow uncontrollably and unevenly,possibly to the extent where at least a proportion of the microbubblesmay cause potentially dangerous embolisation of, for example, themyocardial vasculature and brain (see e.g. Schwarz, Advances inEcho-Contrast [1994(3)], pp. 48-49).

It has also been found that administration of phase shift colloids alonemay not lead to reliable or consistent in vivo volatilisation of thedispersed phase to generate gas or vapour microbubbles. Grayburn et al.in J. Am. Coll. Cardiol. 26(5) [1995], pp. 1340-1347 suggest thatpreactivation of perfluoropentane emulsions may be required to achievemyocardial opacification in dogs at effective imaging doses low enoughto avoid haemodynamic side effects. An activation technique for suchcolloidal dispersions, involving application of hypobaric forcesthereto, is described in WO-A-9640282; typically this involves partiallyfilling a syringe with the emulsion and subsequently forciblywithdrawing and then releasing the plunger of the syringe to generate atransient pressure change which causes formation of gas microbubbleswithin the emulsion. This is an inherently somewhat cumbersome techniquewhich may fail to give consistent levels of activation.

It is stated in U.S. Pat. No. 5,536,489 that emulsions ofwater-insoluble gas-forming chemicals such as perfluoropentane may beused as contrast agents for site-specific imaging, the emulsions onlygenerating a significant number of image-enhancing gas microbubbles uponapplication of ultrasonic energy to a specific location in the bodywhich it is desired to image. Our own research has shown that emulsionsof volatile compounds such as 2-methylbutane or perfluoropentane give nodetectable echo enhancement either in vitro or in vivo whenultrasonicated at energy levels which are sufficient to give pronouncedcontrast effects using two component contrast agents in accordance withthe present invention.

According to one aspect of the invention there is provided a combinedpreparation for simultaneous, separate or sequential use as a contrastagent in ultrasound imaging, said preparation comprising:

-   -   i) an injectable aqueous medium having gas dispersed therein;        and    -   ii) a composition comprising a diffusible component capable of        diffusion in vivo into said dispersed gas so as at least        transiently to increase the size thereof.

According to a further aspect of the invention there is provided amethod of generating enhanced images of a human or non-human animalsubject which comprises the steps of:

-   -   i) injecting a physiologically acceptable aqueous medium having        gas dispersed therein into the vascular system of said subject;    -   ii) before, during or after injection of said aqueous medium        administering to said subject a composition comprising a        diffusible component capable of diffusion in vivo into said        dispersed gas so as at least transiently to increase the size        thereof; and    -   iii) generating an ultrasound image of at least a part of said        subject.

This method according to the invention may advantageously be employed invisualising tissue perfusion in a subject, the increase in size of thedispersed gas being utilised to effect enrichment or temporary retentionof gas in the microvasculature of such tissue, thereby enhancing itsechogenicity.

Any biocompatible gas may be present in the gas dispersion, the term“gas” as used herein including any substances (including mixtures) atleast partially, e.g. substantially or completely in gaseous (includingvapour) form at the normal human body temperature of 37ΕC. The gas maythus, for example, comprise air; nitrogen; oxygen; carbon dioxide;hydrogen; an inert gas such as helium, argon, xenon or krypton; asulphur fluoride such as sulphur hexafluoride, disulphur decafluoride ortrifluoromethylsulphur pentafluoride; selenium hexafluoride; anoptionally halogenated silane such as methylsilane or dimethylsilane; alow molecular weight hydrocarbon (e.g. containing up to 7 carbon atoms),for example an alkane such as methane, ethane, a propane, a butane or apentane, a cycloalkane such as cyclopropane, cyclobutane orcyclopentane, an alkene such as ethylene, propene, propadiene or abutene, or an alkyne such as acetylene or propyne; an ether such asdimethyl ether; a ketone; an ester; a halogenated low molecular weighthydrocarbon (e.g. containing up to 7 carbon atoms); or a mixture of anyof the foregoing. Advantageously at least some of the halogen atoms inhalogenated gases are fluorine atoms; thus biocompatible halogenatedhydrocarbon gases may, for example, be selected frombromochlorodifluoromethane, chlorodifluoromethane,dichlorodifluoromethane, bromotrifluoromethane, chlorotrifluoromethane,chloropentafluoroethane, dichlorotetrafluoroethane,chlorotrifluoroethylene, fluoroethylene, ethylfluoride,1,1-difluoroethane and perfluorocarbons. Representative perfluorocarbonsinclude perfluoroalkanes such as perfluoromethane, perfluoroethane,perfluoropropanes, perfluorobutanes (e.g. perfluoro-n-butane, optionallyin admixture with other isomers such as perfluoro-iso-butane),perfluoropentanes, perfluorohexanes or perfluoroheptanes;perfluoroalkenes such as perfluoropropene, perfluorobutenes (e.g.perfluorobut-2-ene), perfluorobutadiene, perfluoropentenes (e.g.perfluoropent-1-ene) or perfluoro-4-methylpent-2-ene; perfluoroalkynessuch as perfluorobut-2-yne; and perfluorocycloalkanes such asperfluorocyclobutane, perfluoromethylcyclobutane,perfluorodimethylcyclobutanes, perfluorotrimethyl-cyclobutanes,perfluorocyclopentane, perfluoromethyl-cyclopentane,perfluorodimethylcyclopentanes, perfluorocyclohexane,perfluoromethylcyclohexane or perfluorocycloheptane. Other halogenatedgases include methyl chloride, fluorinated (e.g. perfluorinated) ketonessuch as perfluoroacetone and fluorinated (e.g. perfluorinated) etherssuch as perfluorodiethyl ether. The use of perfluorinated gases, forexample sulphur hexafluoride and perfluorocarbons such asperfluoropropane, perfluorobutanes, perfluoropentanes andperfluorohexanes, may be particularly advantageous in view of therecognised high stability in the bloodstream of microbubbles containingsuch gases. Other gases with physicochemical characteristics which causethem to form highly stable microbubbles in the bloodstream may likewisebe useful.

The dispersed gas may be administered in any convenient form, forexample using any appropriate gas-containing ultrasound contrast agentformulation as the gas-containing composition. Representative examplesof such formulations include microbubbles of gas stabilised (e.g. atleast partially encapsulated) by a coalescence-resistant surfacemembrane (for example gelatin, e.g. as described in WO-A-8002365), afilmogenic protein (for example an albumin such as human serum albumin,e.g. as described in U.S. Pat. No. 4,718,433, U.S. Pat. No. 4,774,958,U.S. Pat. No. 4,844,882, EP-A-0359246, WO-A-9112823, WO-A-9205806,WO-A-9217213, WO-A-9406477 or WO-A-9501187), a polymer material (forexample a synthetic biodegradable polymer as described in EP-A-0398935,an elastic interfacial synthetic polymer membrane as described inEP-A-0458745, a microparticulate biodegradable polyaldehyde as describedin EP-A-0441468, a microparticulate N-dicarboxylic acid derivative of apolyamino acid-polycyclic imide as described in EP-A-0458079, or abiodegradable polymer as described in WO-A-9317718 or WO-A-9607434), anon-polymeric and non-polymerisable wall-forming material (for exampleas described in WO-A-9521631), or a surfactant (for example apolyoxyethylene-polyoxypropylene block copolymer surfactant such as aPluronic, a polymer surfactant as described in WO-A-9506518, or afilm-forming surfactant such as a phospholipid, e.g. as described inWO-A-9211873, WO-A-9217212, WO-A-9222247, WO-A-9428780, WO-A-9503835 orWO-A-9729783).

Other useful gas-containing contrast agent formulations includegas-containing solid systems, for example microparticles (especiallyaggregates of microparticles) having gas contained therewithin orotherwise associated therewith (for example being adsorbed on thesurface thereof and/or contained within voids, cavities or porestherein, e.g. as described in EP-A-0122624, EP-A-0123235, EP-A-0365467,WO-A-9221382, WO-A-9300930, WO-A-9313802, WO-A-9313808 or WO-A-9313809).It will be appreciated that the echogenicity of such microparticulatecontrast agents may derive directly from the contained/associated gasand/or from gas (e.g. microbubbles) liberated from the solid material(e.g. upon dissolution of the microparticulate structure).

The disclosures of all of the above-described documents relating togas-containing contrast agent formulations are incorporated herein byreference.

Gas microbubbles and other gas-containing materials such asmicroparticles preferably have an initial average size not exceeding 10μm (e.g. of 7 μm or less) in order to permit their free passage throughthe pulmonary system following administration, e.g. by intravenousinjection. However, larger microbubbles may be employed where, forexample, these contain a mixture of one or more relatively blood-solubleor otherwise diffusible gases such as air, oxygen, nitrogen or carbondioxide with one or more substantially insoluble and non-diffusiblegases such as perfluorocarbons. Outward diffusion of thesoluble/diffusible gas content following administration will cause suchmicrobubbles rapidly to shrink to a size which will be determined by theamount of insoluble/non-diffusible gas present and which may be selectedto permit passage of the resulting microbubbles through the lungcapillaries of the pulmonary system.

Since dispersed gas administered in accordance with the invention iscaused to grow in vivo through interaction with diffusible component,the minimum size of the microbubbles, solid-associated gas etc. asadministered may be substantially lower than the size normally thoughtnecessary to provide significant interaction with ultrasound (typicallyca. 1-5 μm at conventionally-employed imaging frequencies); thedispersed gas moieties may therefore have sizes as low as, for example,1 nm or below. The invention may accordingly permit use ofgas-containing compositions which have not hitherto been proposed foruse as ultrasound contrast agents, e.g. because of the low size of thedispersed gas moieties.

Where phospholipid-containing compositions are employed in accordancewith the invention, e.g. in the form of phospholipid-stabilised gasmicrobubbles, representative examples of useful phospholipids includelecithins (i.e. phosphatidylcholines), for example natural lecithinssuch as egg yolk lecithin or soya bean lecithin, semisynthetic (e.g.partially or fully hydrogenated) lecithins and synthetic lecithins suchas dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine ordistearoylphosphatidylcholine; phosphatidic acids;phosphatidylethanolamines; phosphatidylserines; phosphatidylglycerols;phosphatidylinositols; cardiolipins; sphingomyelins; fluorinatedanalogues of any of the foregoing; mixtures of any of the foregoing andmixtures with other lipids such as cholesterol. The use of phospholipidspredominantly (e.g. at least 75%) comprising molecules individuallybearing net overall charge, e.g. negative charge, for example as innaturally occurring (e.g. soya bean or egg yolk derived), semisynthetic(e.g. partially or fully hydrogenated) and syntheticphosphatidylserines, phosphatidylglycerols, phosphatidylinositols,phosphatidic acids and/or cardiolipins, for example as described inWO-A-9729783, may be particularly advantageous.

Representative examples of gas-containing microparticulate materialswhich may be useful in accordance with the invention includecarbohydrates (for example hexoses such as glucose, fructose orgalactose; disaccharides such as sucrose, lactose or maltose; pentosessuch as arabinose, xylose or ribose; □-, □- and □-cyclodextrins;polysaccharides such as starch, hydroxyethyl starch, amylose,amylopectin, glycogen, inulin, pulullan, dextran, carboxymethyl dextran,dextran phosphate, ketodextran, aminoethyldextran, alginates, chitin,chitosan, hyaluronic acid or heparin; and sugar alcohols, includingalditols such as mannitol or sorbitol), inorganic salts (e.g. sodiumchloride), organic salts (e.g. sodium citrate, sodium acetate or sodiumtartrate), X-ray contrast agents (e.g. any of the commercially availablecarboxylic acid and non-ionic amide contrast agents typically containingat least one 2,4,6-triiodophenyl group having substituents such ascarboxyl, carbamoyl, N-alkylcarbamoyl, N-hydroxyalkylcarbamoyl,acylamino, N-alkylacylamino or acylaminomethyl at the 3- and/or5-positions, as in metrizoic acid, diatrizoic acid, iothalamic acid,ioxaglic acid, iohexol, iopentol, iopamidol, iodixanol, iopromide,metrizamide, iodipamide, meglumine iodipamide, meglumine acetrizoate andmeglumine diatrizoate), and polypeptides and proteins (e.g. gelatin oralbumin such as human serum albumin).

Other gas-containing materials which may be useful in accordance withthe invention include gas-containing material stabilised by metals (e.g.as described in US-A-3674461 or U.S. Pat. No. 3,528,809), gas-containingmaterial stabilised by synthetic polymers (e.g. as described in U.S.Pat. No. 3,975,194 or by Farnand in Powder Technology 22 [1979], pp.11-16), commercially available microspheres of the Expance17 type, e.g.Expancel 551 DE (see e.g. Eur. Plast. News 9(5) [1982], p. 39, NonwovensIndustry [1981], p. 21 and Mat. Plast. Elast. 10 [1980], p. 468),commercially available microspheres of the Ropaque7 type (see e.g. J.Coatings Technol. 55(707) [1983], p. 79), micro- and nano-sizedgas-containing structures such as zeolites, inorganic or organicaerogels, nanosized open void-containing chemical structures such asfullerenes, clathrates or nanotubes (e.g. as described by G. E. Gadd inScience 277 (5328) [1997], pp. 933-936), and naturalsurfactant-stabilised microbubble dispersions (e.g. as described byd'Arrigo in “Stable Gas-in-Liquid Emulsions, Studies in physical andtheoretical chemistry” 40—Elsevier, Amsterdam [1986]).

A wide range of diffusible components may be used in accordance with theinvention, including gases/vapours, volatile liquids, volatile solidsand precursors capable of gas generation, e.g. upon administration, theprincipal requirement being that the component should either have or becapable of generating a sufficient gas or vapour pressure in vivo (e.g.at least 10 torr) so as to be capable of promoting inward diffusion ofgas or vapour molecules into the dispersed gas. It will be appreciatedthat mixtures of two or more diffusible components may if desired beemployed in accordance with the invention; references herein to “thediffusible component” are to be interpreted as including such mixtures.Similarly, references to administration of a diffusible component areintended also to embrace administration of two or more such components,either as mixtures or as plural administrations.

The composition comprising the diffusible component may take anyappropriate form and may be administered by any appropriate method, theroute of administration depending in part on the area of the subjectwhich is to be investigated. Thus, for example, oral administration ofan appropriate composition comprising a diffusible component may beparticularly useful where it is desired to promote temporary retentionof gas in the tissue of the gastrointestinal wall. In representativeembodiments of such applications the gas dispersion may be injectedintravenously in doses similar to those used in echocardiography and thediffusible component may be formulated as an orally administrableemulsion, e.g. a perfluorocarbon-in-water emulsion as described infurther detail hereinafter, for example being used at a dose of 0.2-1.0μl perfluorocarbon/kg. Following administration and distribution of thetwo compositions, growth of the gas dispersion in the capillary bloodpool in the gastric or intestinal wall may enhance contour contrast fromthese regions. It will be appreciated that the reverse combination of anorally administrable gas dispersion and intravenously injectablediffusible component may be useful in providing contour contrast fromthe inner wall or mucosa of the gastrointestinal system.

It may be advantageous when using such orally administrable gasdispersion or diffusible component compositions to incorporate chemicalgroups or substances which promote adhesion to the wall of thegastrointestinal tract, for example by admixture with the composition orby attachment to a component thereof, e.g. a surfactant or otherstsbilising moiety, since this may stimulate growth of the dispersed gasphase by enhancing its contact with the diffusible component. Examplesof such adhesion-promoting groups/substances have previously beendescribed in relation to, for example, gastrointestinal X-ray contrastagents, and include acrylic esters as described in WO-A-9722365,iodophenol sulphonate esters as described in U.S. Pat. No. 5,468,466 andiodinated phenyl esters as described in U.S. Pat. No. 5,260,049.

Inhalation of a suitably volatile diffusible component may, for example,be used to promote growth of the administered gas dispersion immediatelyfollowing its passage through the lung capillaries, e.g. so that the gasthen becomes temporarily retained in the capillaries of the myocardium.In such embodiments growth of the dispersed gas may be further increasedby raising the lung pressure of the diffusible component, e.g. by anexcess of up to 0.5 bar, for example by using a respirator or by havingthe subject exhale against a resistance.

Intramuscular or subcutaneous injection of appropriately formulateddiffusible component compositions, e.g. incorporating a physiologicallyacceptable carrier liquid, may, for example, advantageously be employedwhere it is desired specifically to limit the effect of the component toa particular target area of the subject. One example of a compositionfor subcutaneous injection comprises nanoparticles such as are used forlymph angiography. Subcutaneously injected diffusible component may betaken up by the lymph system, where it may cause growth of anintravenously injected gas dispersion, thereby facilitating imaging oflymph nodes. The reverse combination of a subcutaneously injected gasdiapersion and intravenously injected diffusible component may similarlybe employed.

Intravenous injection of appropriately formulated diffusible componentcompositions, e.g. incorporating a physiologically acceptable carrierliquid, permits considerable versatility in operation of the inventionsince, as discussed in greater detail hereinafter, the constituents ofthe gas dispersion and diffusible component compositions may be selectedto control parameters such as the onset and rate of growth of thedispersed gas and thus the parts of the body in which tissueechogenicity may be enhanced by temporary retention of gas, for examplein the microvasculature thereof.

Appropriate topical formulations may be applied cutaneously so as topromote transcutaneous absorption of the diffusible component. Suchadministration may have applications in imaging and/or therapy of theskin, subcutis and adjacent regions and organs, for example in targetingthe peripheral circulation of body extremities such as legs.

Diffusible components for administration orally or by injection may, forexample, be formulated as solutions in or mixtures with water and/or oneor more water-miscible and physiologically acceptable organic solvents,such as ethanol, glycerol or polyethylene glycol; dispersions in anaqueous medium, for example as the oil phase or a constituent of the oilphase of an oil-in-water emulsion; microemulsions, i.e. systems in whichthe substance is effectively dissolved in the hydrophobic interiors ofsurfactant micelles present in an aqueous medium; or in association withmicroparticles or nanoparticles dispersed in an appropriate carrierliquid, for example being adsorbed on microparticle or nanoparticlesurfaces and/or contained within voids, cavities or pores ofmicroparticles or nanoparticles, or encapsulated within microcapsules.

Where a diffusible component is to be administered as a solution, thepartial pressure derived therefrom in vivo will depend on theconcentration of the component, e.g. in the blood stream, and thecorresponding pressure of pure component material, for example inaccordance with Raoult's law in a system approaching ideality. Thus ifthe component has low water solubility it is desirable that it shouldhave a sufficient vapour pressure in pure form at normal bodytemperature, e.g. at least 50 torr, preferably at least 100 torr.Examples of relatively water-insoluble components with high vapourpressures include gases such as those listed hereinbefore as possiblemicrobubble gases.

Representative examples of more highly water-soluble/water-misciblediffusible components, which may therefore exhibit lower vapourpressures at body temperature, include aliphatic ethers such as ethylmethyl ether or methyl propyl ether; aliphatic esters such as methylacetate, methyl formate or ethyl formate; aliphatic ketones such asacetone; aliphatic amides such as N,N-dimethylformamide orN,N-dimethylacetamide; and aliphatic nitriles such as acetonitrile.

It may, however, be preferred to employ a substantially water-immisciblediffusible component formulated as an emulsion (i.e. a stabilisedsuspension) in an appropriate aqueous medium, since in such systems thevapour pressure in the aqueous phase of the diffusible component will besubstantially equal to that of pure component material, even in verydilute emulsions. In such embodiments the diffusible component may, forexample, be formulated as part of a proprietary registeredpharmaceutical emulsion, such as Intralipid7 (Pharmacia).

The diffusible component in such emulsions is advantageously a liquid atprocessing and storage temperature, which may for example be as low as−10ΕC if the aqueous phase contains appropriate antifreeze material,while being a gas or exhibiting a substantial vapour pressure at bodytemperature. Appropriate compounds may, for example, be selected fromthe various lists of emulsifiable low boiling liquids given in theaforementioned WO-A-9416379, the contents of which are incorporatedherein by reference. Specific examples of emulsifiable diffusiblecomponents include aliphatic ethers such as diethyl ether; polycyclicoils or alcohols such as menthol, camphor or eucalyptol; heterocycliccompounds such as furan or dioxane; aliphatic hydrocarbons, which may besaturated or unsaturated and straight chained or branched, e.g. as inn-butane, n-pentane, 2-methylpropane, 2-methylbutane,2,2-dimethylpropane, 2,2-dimethylbutane, 2,3-dimethylbutane, 1-butene,2-butene, 2-methylpropene, 1,2-butadiene, 1,3-butadiene,2-methyl-1-butene, 2-methyl-2-butene, isoprene, 1-pentene,1,3-pentadiene, 1,4-pentadiene, butenyne, 1-butyne, 2-butyne or1,3-butadiyne; cycloaliphatic hydrocarbons such as cyclobutane,cyclobutene, methylcyclopropane or cyclopentane; and halogenated lowmolecular weight hydrocarbons (e.g. containing up to 7 carbon atoms).Representative halogenated hydrocarbons include dichloromethane, methylbromide, 1,2-dichloroethylene, 1,1-dichloroethane, 1-bromoethylene,1-chloroethylene, ethyl bromide, ethyl chloride, 1-chloropropene,3-chloropropene, 1-chloropropane, 2-chloropropane and t-butyl chloride.Advantageously at least some of the halogen atoms are fluorine atoms,for example as in dichlorofluoromethane, trichlorofluoromethane,1,2-dichloro-1,2-difluoroethane, 1,2-dichloro-1,1,2,2-tetrafluoroethane,1,1,2-trichloro-1,2,2-trifluoroethane,2-bromo-2-chloro-1,1,1-trifluoroethane, 2-chloro-1,1,2-trifluoroethyldifluoromethyl ether, 1-chloro-2,2,2-trifluoroethyl difluoromethylether, partially fluorinated alkanes (e.g. pentafluoropropanes such as1H,1H,3H-pentafluoropropane, hexafluorobutanes, nonafluorobutanes suchas 2H-nonafluoro-t-butane, and decafluoropentanes such as2H,3H-decafluoropentane), partially fluorinated alkenes (e.g.heptafluoropentenes such as 1H,1H,2H-heptafluoropent-1-ene, andnonafluorohexenes such as 1H,1H,2H-nonafluorohex-1-ene), fluorinatedethers (e.g. 2,2,3,3,3-pentafluoropropyl methyl ether or2,2,3,3,3-pentafluoropropyl difluoromethyl ether) and, more preferably,perfluorocarbons. Examples of perfluorocarbons include perfluoroalkanessuch as perfluorobutanes, perfluoropentanes, perfluorohexanes (e.g.perfluoro-2-methylpentane), perfluoroheptanes, perfluorooctanes,perfluorononanes and perfluorodecanes; perfluorocycloalkanes such asperfluorocyclobutane, perfluorodimethyl-cyclobutanes,perfluorocyclopentane and perfluoromethylcyclopentane; perfluoroalkenessuch as perfluorobutenes (e.g. perfluorobut-2-ene orperfluorobuta-1,3-diene), perfluoropentenes (e.g. perfluoropent-1-ene)and perfluorohexenes (e.g. perfluoro-2-methylpent-2-ene orperfluoro-4-methylpent-2-ene); perfluorocycloalkenes such asperfluorocyclopentene or perfluorocyclopentadiene; and perfluorinatedalcohols such as perfluoro-t-butanol.

Such emulsions may also contain at least one surfactant in order tostabilise the dispersion; this may be the same as or different from anysurfactant(s) used to stabilise the gas dispersion. The nature of anysuch surfactant may significantly affect factors such as the rate ofgrowth of the dispersed gas phase. In general a wide range ofsurfactants may be useful, for example selected from the extensive listsgiven in EP-A-0727225, the contents of which are incorporated herein byreference. Representative examples of useful surfactants include fattyacids (e.g. straight chain saturated or unsaturated fatty acids, forexample containing 10-20 carbon atoms) and carbohydrate and triglycerideesters thereof, phospholipids (e.g. lecithin), fluorine-containingphospholipids, proteins (e.g. albumins such as human serum albumin),polyethylene glycols, and block copolymer surfactants (e.g.polyoxyethylene-polyoxypropylene block copolymers such as Pluronics,extended polymers such as acyloxyacyl polyethylene glycols, for examplepolyethyleneglycol methyl ether 16-hexadecanoyloxy-hexadecanoate, e.g.wherein the polyethylene glycol moiety has a molecular weight of 2300,5000 or 10000), and fluorine-containing surfactants (e.g. as marketedunder the trade names Zonyl and Fluorad, or as described inWO-A-9639197, the contents of which are incorporated herein byreference). Particularly useful surfactants include phospholipidscomprising molecules with net overall negative charge, such as naturallyoccurring (e.g. soya bean or egg yolk derived), semisynthetic (e.g.partially or fully hydrogenated) and synthetic phosphatidylserines,phosphatidylglycerols, phosphatidylinositols, phosphatidic acids and/orcardiolipins.

The droplet size of the dispersed diffusible component in emulsionsintended for intravenous injection should preferably be less than 10 μm,e.g. less than 7 μm, and greater than 0.1 μm in order to facilitateunimpeded passage through the pulmonary system.

As noted above, water-immiscible diffusible components may also beformulated as microemulsions. Such systems are advantageous by virtue oftheir thermodynamic stability and the fact that the diffusible componentis in practice uniformly distributed throughout the aqueous phase;microemulsions therefore have the appearance of solutions but mayexhibit the properties of emulsions as regards the partial pressure ofthe dispersed phase.

Gas precursors which may be used include any biocompatible componentscapable of gas generation in vivo, i.e. at body temperature andphysiological pH. Representative examples include inorganic and organiccarbonates and bicarbonates, and nitrogen-generating substances such aspyrazolines, pyrazoles, triazolines, diazoketones, diazonium salts,tetrazoles and azides. It will be appreciated that in such systems it isthe ultimately generated gas which is the actual diffusible component.

In order to ensure maximum volatilisation of the diffusible componentfollowing administration and to enhance growth of the dispersed gas,both of which are endothermic processes, it may be advantageous tomanipulate the temperature of the solution or suspension of thediffusible component and/or the gas dispersion prior to administrationand/or to incorporate exothermically reactive constituents therein; theuse of such constituents which react exothermically under the influenceof ultrasound radiation may be particularly advantageous.

Growth of the dispersed gas phase in vivo may, for example, beaccompanied by expansion of any encapsulating material (where this hassufficient flexibility) and/or by abstraction of excess surfactant fromthe administered material to the growing gas-liquid interfaces. It isalso possible, however, that stretching of the encapsulating materialand/or interaction of the material with ultrasound may substantiallyincrease its porosity. Whereas such disruption of encapsulating materialhas hitherto in many cases been found to lead to rapid loss ofechogenicity through outward diffusion and dissolution of the gasthereby exposed, we have found that when using contrast agentpreparations in accordance with the present invention, the exposed gasexhibits substantially stability. Whilst not wishing to be bound bytheoretical calculations, we believe that the exposed gas, e.g. in theform of liberated microbubbles, may be stabilised, e.g. against collapseof the microbubbles, by the supersaturated environment generated by thediffusible component, which provides an inward pressure gradient tocounteract the outward diffusive tendency of the microbubble gas. Theexposed gas surface, by virtue of the substantial absence ofencapsulating material, may cause the contrast agent preparation toexhibit exceptionally favourable acoustic properties as evidenced byhigh backscatter and low energy absorption (e.g. as expressed by highbackscatter: attenuation ratios); this echogenic effect may continue fora significant period, even during continuing ultrasound irradiation.

The stabilising effect of coadministered diffusible component maytherefore be used to great advantage to enhance both the duration andmagnitude of the echogenicity of existing gas-containing contrast agentformulations in cases where these parameters may be insufficient whenthe contrast agent composition is administered alone. Thus, for example,the duration of effect of albumin-based contrast agents is oftenseverely limited by collapse of the encapsulating albumin material,either as a result of systolic pressure changes in the heart or venoussystem or as a consequence of ultrasound irradiation, but may besubstantially enhanced by coadministration with a diffusible componentin accordance with the present invention.

In a representative embodiment of the method of the invention acomposition comprising a gas dispersion and a composition comprising adiffusible component suspension are selected such that at least aproportion of the dispersed gas passes through the lungs and thenundergoes rapid growth following passage from the lungs through inwarddiffusion of the diffusible component, so as temporarily to be retainedin the myocardium and thereby permit ultrasonic visualisation ofmyocardial perfusion. As the concentration of volatile diffusiblecomponent in the bloodstream falls away, e.g. as the component iscleared from the blood, for example by removal through the lungs andexhalation by the subject, by metabolism or by redistribution to othertissues, the diffusible component will typically diffuse out of thedispersed gas, which will therefore shrink towards its initial smallersize, and ultimately once more becoming free flowing in the bloodstream,typically being removed therefrom by the reticuloendothelial system.This pattern of a substantial transient increase in echogenicityfollowed by disappearance of contrast effect is markedly different fromany echogenic properties exhibited by either of the two compositionswhen administered alone. It will be appreciated from the foregoing thatcontrol of the duration of retention of the dispersed gas may thereforebe achieved by appropriate adjustment of the dose and/or formulation ofthe diffusible component.

Other capillary systems, such as but not limited to those of the kidney,liver, spleen, thyroid, skeletal muscle, breast and penis, may similarlybe imaged.

It will be appreciated that factors such as the rate and/or extent ofgrowth of the dispersed gas may in general be controlled by appropriateselection of the gas and any encapsulating stabilising material and,more particularly, the nature of the diffusible component and the mannerin which it is formulated, including the nature of any surfactantemployed and the size of the dispersed phase droplets where thecomponent is formulated as an emulsion; in this last context, for agiven amount of emulsified diffusible component, a reduction in dropletsize may enhance the rate of transfer of diffusible component relativeto that from larger droplets since more rapid release may occur fromsmaller droplets having higher surface area:volume ratios. Otherparameters permitting control include the relative amounts in which thetwo compositions are administered and, where these are administeredseparately, the order of administration, the time interval between thetwo administrations, and possible spatial separation of the twoadministrations. In this last respect it will be appreciated that theinherent diffusivity of the diffusible component may permit itsapplication to different parts of the body in a wide variety of ways,for example by inhalation, cutaneously, subcutaneously, intravenously,intramuscularly or orally, whereas the available forms of administrationfor the dispersed gas may be somewhat more limited.

Particularly important parameters with regard to the diffusiblecomponent are its solubility in water/blood and its diffusibility (e.g.as expressed by its diffusion constants), which will determine its rateof transport through the carrier liquid or blood, and its permeabilitythrough any membrane encapsulating the dispersed gas. The pressuregenerated by the diffusible component in vivo will also affects its rateof diffusion into the dispersed gas, as will its concentration. Thus, inaccordance with Fick's law, the concentration gradient of diffusiblecomponent relative to the distance between, for example, individual gasmicrobubbles and emulsion droplets, together with the diffusioncoefficient of the diffusible substance in the surrounding liquidmedium, will determine the rate of transfer by simple diffusion; theconcentration gradient is determined by the solubility of the diffusiblecomponent in the surrounding medium and the distance between individualgas microbubbles and emulsion droplets.

The effective rate of transport of the diffusible component may, ifdesired, be controlled by adjusting the viscosity of the dispersed gasphase composition and/or the diffusible component composition, forexample by incorporating one or more biocompatible viscosity enhancerssuch as X-ray contrast agents, polyethylene glycols, carbohydrates,proteins, polymers or alcohols into the formulation. It may, forexample, be advantageous to coinject the two compositions as arelatively high volume bolus (e.g. having a volume of at least 20 ml inthe case of a 70 kg human subject), since this will delay completemixing of the constituents with blood (and thus the onset of growth ofthe dispersed gas) until after entry into the right ventricle of theheart and the lung capillaries. The delay in growth of the dispersed gasmay be maximised by employing carrier liquid which is undersaturatedwith respect to gases and any other diffusible components ashereinbefore defined, e.g. as a result of being cooled.

As noted above, transport mechanisms other than diffusion may beinvolved in operation of the invention. Thus, for example, transport mayalso occur through hydrodynamic flow within the surrounding liquidmedium; this may be important in vessels and capillaries where highshear rate flow may occur. Transport of diffusible component to thedispersed gas may also occur as a result of collision or near-collisionprocesses, e.g. between gas microbubbles and emulsion droplets, forexample leading to adsorption of diffusible component at the microbubblesurface and/or penetration of diffusible component into the microbubble,i.e. a form of coalescence. In such cases the diffusion coefficient andsolubility of the diffusible component have a minimal effect on the rateof transfer, the particle size of the diffusible component (e.g. thedroplet size where this is formulated as an emulsion) and the collisionfrequency between microbubbles and droplets being the principal factorscontrolling the rate and extent of microbubble growth. Thus, forexample, for a given amount of emulsified diffusible component, areduction in droplet size will lead to an increased overall number ofdroplets and so may enhance the rate of transfer by reducing the meaninterparticle distance between the gas microbubbles and emulsiondroplets and thus increasing the probability of collision and/orcoalescence. It will be appreciated that the rate of transfersproceeding through collision processes may be markedly enhanced ifadditional oscillatory movement is imparted to the gas microbubbles andemulsion droplets of the diffusible component through application ofultrasonic energy. The kinetics of collision processes induced by suchultrasonic energy may differ from the kinetics for transport ofdiffusible component in carrier liquid and/or blood, for example in thatspecific energy levels may be necessary to initiate coalescence ofcolliding gas microbubbles and emulsion droplets. Accordingly it may beadvantageous to select the size and therefore the mass of the emulsiondroplets so that they generate sufficient collisional force with theoscillating microbubbles to induce coalescence.

As also noted above, the permeability of any material encapsulating thedispersed gas phase is a parameter which may affect the rate of growthof the gas phase, and it may therefore be desirable to select adiffusible component which readily permeates any such encapsulatingmaterial (which may, for example, be a polymer or surfactant membrane,e.g. a monolayer or one or more bilayers of a membrane-formingsurfactant such as a phospholipid). We have found, however, thatsubstantially impermeable encapsulating material may also be used, sinceit appears that sonication, including sonication at lower and higherfrequencies than normally used in medical ultrasound imaging (e.g. inthe range 10 Hz to 1 GHz, preferably between 1 kHz and 10 MHz) and witheither continuous radiation or simple or complex pulse patterns, ofcombined contrast agent preparations administered according to theinvention may itself promote or enhance growth of the dispersed gas.Such growth may, for example, be induced by the ultrasound irradiationused to effect an investigation or by preliminary localised irradiation,e.g. serving to effect temporary retention of gas in themicrovasculature of a particular target organ. Alternatively, activationof growth of the dispersed gas may be induced by aplication ofsufficient amounts of other forms of energy, for exaple shaking,vibration, an electric field, radiation or particle bombardment, e.g.with neutral particles, ions or electrons.

Whilst we do not wish to be bound by theoretical considerations it maybe that ultrasonication at least transiently modifies the permeabilityof the encapsulating material, the diffusibility of the diffusiblecomponent in the surrounding liquid phase and/or the frequency ofcollisions between emulsion droplets and the encapsulated microbubbles.Since the effect may be observed using extremely short ultrasound pulses(e.g. with durations of ca. 0.3 μs in B-mode imaging or ca. 2 μs inDoppler or second harmonic imaging) it seems unlikely to be an exampleof rectified diffusion, in which ongoing ultrasound irradiation producesa steady increase in the equilibrium radii of gas bubbles (see Leighton,E. G.—“The Acoustic Bubble”, Academic Press [1994], p. 379), and it maybe that the ultrasound pulses disrupt the encapsulating membrane and soenhance growth of the dispersed gas through inward diffusion ofdiffusible component into the thus-exposed gas phase.

If desired, either the dispersed gas or the diffusible component maycomprise an azeotropic mixture or may be selected so that an azeotropicmixture is formed in vivo as the diffusible component mixes with thedispersed gas. Such azeotrope formation may, for example, be usedeffectively to enhance the volatility of relatively high molecularweight compounds, e.g. halogenated hydrocarbons such as fluorocarbons(including perfluorocarbons) which under standard conditions are liquidat the normal human body temperature of 37ΕC, such that they may beadministered in gaseous form at this temperature. This has substantialbenefits as regards the effective echogenic lifetime in vivo of contrastagents containing such azeotropic mixtures since it is known thatparameters such as the water solubility, fat solubility, diffusibilityand pressure resistivity of compounds such as fluorocarbons decreasewith increasing molecular weight.

In general, the recognised natural resistance of azeotropic mixtures toseparation of their constituents will enhance the stability of contrastagent components containing the same, both during preparation, storageand handling and following administration.

Azeotropic mixtures useful in accordance with the invention may, forexample, be selected by reference to literature relating to azeotropes,by experimental investigation and/or by theoretical predictions, e.g. asdescribed by Tanaka in Fluid Phase Equilibria 24 (1985), pp. 187-203, byKittel, C. and Kroemer, H. in Chapter 10 of Thermal Physics (W.H.Freeman & Co., New York, USA, 1980) or by Hemmer, P. C. in Chapters16-22 of Statistisk Mekanikk (Tapir, Trondheim, Norway, 1970), thecontents of which are incorporated herein by reference.

One literature example of an azeotrope which effectively reduces theboiling point of the higher molecular weight component to below normalbody temperature is the 57:43 w/w mixture of1,1,2-trichloro-1,2,2-trifluoromethane (b.p. 47.6ΕC) and1,2-difluoro-methane (b.p. 29.6ΕC) described in U.S. Pat. No. 4,055,049as having an azeotropic boiling point of 24.9ΕC. Other examples ofhalocarbon-containing azeotropic mixtures are disclosed in EP-A-0783017,U.S. Pat. No. 5,599,783, U.S. Pat. No. 5,605,647, U.S. Pat. No.5,605,882, U.S. Pat. No. 5,607,616, U.S. Pat. No. 5,607,912, U.S. Pat.No. 5,611,210, U.S. Pat. No. 5,614,565 and U.S. Pat. No. 5,616,821, thecontents of which are incorporated herein by reference.

Simons et al. in J. Chem. Phys. 18(3) (1950), pp. 335-346 report thatmixtures of perfluoro-n-pentane (b.p. 29ΕC) and n-pentane (b.p. 36ΕC)exhibit a large positive deviation from Raoult's law; the effect is mostpronounced for approximately equimolar mixtures. In practice the boilingpoint of the azeotropic mixture has been found to be about 22ΕC or less.Mixtures of perfluorocarbons and unsubstituted hydrocarbons may ingeneral exhibit useful azeotropic properties; strong azeotropic effectshave been observed for mixtures of such components having substantiallysimilar boiling points. Examples of other perfluorocarbon:hydrocarbonazeotropes include mixtures of perfluoro-n-hexane (b.p. 59ΕC) andn-pentane, where the azeotrope has a boiling point between roomtemperature and 35ΕC, and of perfluoro-4-methylpent-2-ene (b.p. 49ΕC)and n-pentane, where the azeotrope has a boiling point of approximately25ΕC.

Other potentially useful azeotropic mixtures include mixtures ofhalothane and diethyl ether and mixtures of two or more fluorinatedgases, for example perfluoropropane and fluoroethane, perfluoropropaneand 1,1,1-trifluoroethane, or perfluoroethane and difluoromethane.

It is known that fluorinated gases such as perfluoroethane may formazeotropes with carbon dioxide (see e.g. WO-A-9502652). Accordingly,administration of contrast agents containing such gases may lead to invivo formation of ternary or higher azeotropes with blood gases such ascarbon dioxide, thereby further enhancing the stability of the dispersedgas.

Where the two compositions of combined contrast agent preparationsaccording to the invention are to be administered simultaneously theymay, for example, be injected from separate syringes via suitablecoupling means or may be premixed, preferably under controlledconditions such that premature microbubble growth is avoided.

Compositions intended for mixing prior to simultaneous administrationmay advantageously be stored in appropriate dual or multi-chamberdevices. Thus, for example, the gas dispersion composition or a driedprecursor therefor [e.g. comprising a lyophilised residue of asuspension of gas microbubbles in an amphiphilic material-containingaqueous medium, particularly wherein the amphiphilic material consistsessentially of phospholipid predominantly (e.g. at least 75%, preferablysubstantially completely) comprising molecules which individually havean overall net (e.g. negative) charge] may be contained in a firstchamber such as a vial, to which a syringe containing the diffusiblecomponent composition is sealing connected; the syringe outlet isclosed, e.g. with a membrane or plug, to avoid premature mixing.Operation of the syringe plunger ruptures the membrane and causes thediffusible component composition to mix with the gas dispersioncomponent or to mix with and reconstitute a precursor therefor;following any necessary or desired shaking and/or dilution, the mixturemay be withdrawn (e.g. by syringe) and administered.

Alternatively the two compositions may be stored within a single sealedvial or syringe, being separated by, for example, a membrane or plug; anoverpressure of gas or vapour may be applied to either or bothcompositions. Rupture of the membrane or plug, e.g. by insertion of ahypodermic needle into the vial, leads to mixing of the compositions;this may if desired be enhanced by hand-shaking, whereafter the mixturemay be withdrawn and administered. Other embodiments, for example inwhich a vial containing a dried precursor for the gas dispersioncomposition is fitted with a first syringe containing a redispersionfluid for said precursor and a second syringe containing the diffusiblecomponent composition, or in which a vial containing membrane-separateddiffusible component composition and dried precursor for the gasdispersion composition is fitted with a syringe containing redispersionfluid for the latter, may similarly be used.

In embodiments of the invention in which the gas dispersion compositionand diffusible component composition are mixed prior to administration,either at the manufacturing stage or subsequently, the mixture willtypically be stored at elevated pressure or reduced temperature suchthat the pressure of the diffusible component is insufficient to providegrowth of the dispersed gas. Activation of growth of the dispersed gasmay be induced simply by release of excess pressure or by the heating tobody temperature which will follow administration of the mixture, or itmay if desired be brought about by preheating the mixture immediatelybefore administration.

In embodiments of the invention in which the gas dispersion compositionand diffusible component composition are administered separately, thetiming between the two administrations may be used to influence the areaof the body in which growth of the dispersed gas phase predominantlyoccurs. Thus, for example, the diffusible component may be injectedfirst and allowed to concentrate in the liver, thereby enhancing imagingof that organ upon subsequent injection of the gas dispersion. Where thestability of the gas dispersion permits, this may likewise be injectedfirst and allowed to concentrate in the liver, with the diffusiblecomponent then being administered to enhance the echogenicity thereof.

Imaging modalities which may be used in accordance with the inventioninclude two- and three-dimensional imaging techniques such as B-modeimaging (for example using the time-varying amplitude of the signalenvelope generated from the fundamental frequency of the emittedultrasound pulse, from sub-harmonics or higher harmonics thereof or fromsum or difference frequencies derived from the emitted pulse and suchharmonics, images generated from the fundamental frequency or the secondharmonic thereof being preferred), colour Doppler imaging, Doppleramplitude imaging and combinations of these last two techniques with anyof the other modalities described above. For a given dose of the gasdispersion and diffusible component compositions, the use of colourDoppler imaging ultrasound to induce growth of the dispersed gas hasbeen found to give stronger contrast effects during subsequent B-modeimaging, possibly as a result of the higher ultrasound intensitiesemployed. To reduce the effects of movement, successive images oftissues such as the heart or kidney may be collected with the aid ofsuitable synchronisation techniques (e.g. gating to the ECG orrespiratory movement of the subject). Measurement of changes inresonance frequency or frequency absorption which accompany growth ofthe dispersed gas may also usefully be made to detect the contrastagent.

It will be appreciated that the dispersed gas content of combinedcontrast agent preparations according to the invention will tend to betemporarily retained in tissue in concentrations proportional to theregional rate of tissue perfusion. Accordingly, when using ultrasoundimaging modalities such as conventional or harmonic B-mode imaging wherethe display is derived directly from return signal intensities, imagesof such tissue may be interpreted as perfusion maps in which thedisplayed signal intensity is a function of local perfusion. This is incontrast to images obtained using free-flowing contrast agents, wherethe regional concentration of contrast agent and corresponding returnsignal intensity depend on the actual blood content rather than the rateof perfusion of local tissue.

In cardiac studies, where perfusion maps are derived from return signalintensities in accordance with this embodiment of the invention, it maybe advantageous to subject a patient to physical or pharmacologicalstress in order to enhance the distinction, and thus the difference inimage intensities, between normally perfused myocardium and anymyocardial regions supplied by stenotic arteries. As is known fromradionucleide cardiac imaging, such stress induces vasodilatation andincreased blood flow in healthy myocardial tissue, whereas blood flow inunderperfused tissue supplied by a stenotic artery is substantiallyunchanged since the capacity for arteriolar vasodilatation is alreadyexhausted by inherent autoregulation seeking to increase the restrictedblood flow.

The application of stress as physical exercise or pharmacologically byadministration of adrenergic agonists may cause discomfort such as chestpains in patient groups potentially suffering from heart disease, and itis therefore preferable to enhance the perfusion of healthy tissue byadministration of a vasodilating drug, for example selected fromadenosine, dipyridamole, nitroglycerine, isosorbide mononitrate,prazosin, doxazosin, dihydralazine, hydralazine, sodium nitroprusside,pentoxyphylline, amelodipine, felodipine, isradipine, nifedipine,nimodipine, verapamil, diltiazem and nitrous oxide. In the case ofadenosine this may lead to in excess of fourfold increases in coronaryblood flow in healthy myocardial tissue, greatly increasing the uptakeand temporary retention of contrast agents in accordance with theinvention and thus significantly increasing the difference in returnsignal intensities between normal and hypoperfused myocardial tissue.Because an essentially physical entrapment process is involved,retention of contrast agents according to the invention is highlyefficient; this may be compared to the uptake of radionucleide tracerssuch as thallium 201 and technetium sestamibi, which is limited by lowcontact time between tracer and tissue and so may require maintenance ofvasodilatation for the whole period of blood pool distribution for thetracer (e.g. 4-6 minutes for thallium scintigraphy) to ensure optimumeffect. The contrast agents of the invention, on the other hand, do notsuffer such diffusion or transport limitations, and since theirretention in myocardial tissue may also rapidly be terminated, forexample by cessation of growth-generating ultrasound irradiation, theperiod of vasodilatation needed to achieve cardiac perfusion imaging inaccordance with this embodiment of the invention may be very short, forexample less than one minute. This will reduce the duration of anypossible discomfort caused to patients by administration of vasodilatordrugs.

In view of the fact that the required vasodilatation need only be shortlasting, adenosine is a particularly useful vasodilating drug, beingboth an endogenous substance and having a very short-lasting action asevidenced by a blood pool half-life of only 2 seconds. Vasodilatationwill accordingly be most intense in the heart, since the drug will tendto reach more distal tissues in less than pharmacologically activeconcentrations. It will be appreciated that because of this shorthalf-life, repeated injection or infusion of adenosine may be necessaryduring cardiac imaging in accordance with this embodiment of theinvention; by way of example, an initial administration of 150 μg/kg ofadenosine may be made substantially simultaneously with administrationof the contrast agent composition, followed 10 seconds later by slowinjection of a further 150 μg/kg of adenosine, e.g. over a period of 20seconds.

Contrast agent preparations in accordance with the invention mayadvantageously be employed as delivery agents for bioactive moietiessuch as therapeutic drugs (i.e. agents having a beneficial effect on aspecific disease in a living human or non-human animal), particularly totargeted sites. Thus, for example, therapeutic compounds may be presentin the dispersed gas, may be linked to part of an encapsulating wall ormatrix, e.g. through covalent or ionic bonds, if desired through aspacer arm, or may be physically mixed into such encapsulating or matrixmaterial; this last option is particularly applicable where thetherapeutic compound and encapsulating or matrix material have similarpolarities or solubilities.

The controllable growth properties of the dispersed gas may be utilisedto bring about its temporary retention in the microvasculature of atarget region of interest; use of ultrasonic irradiation to inducegrowth and thus retention of the gas and associated therapeutic compoundin a target structure is particularly advantageous. Localised injectionof the gas dispersion composition or, more preferably, the diffusiblecomponent composition, e.g. as hereinbefore described, may also be usedto concentrate growth of the dispersed gas in a target area.

The therapeutic compound, which may if desired be coupled to asite-specific vector having affinity for specific cells, structures orpathological sites, may be released as a result of, for example,stretching or fracture of the encapsulating or matrix material caused bygrowth of the dispersed gas, solubilisation of the encapsulating ormatrix material, or disintegration of microbubbles or microparticles(e.g. induced by ultra-sonication or by a reversal of the concentrationgradient of the diffusible component in the target area). Where atherapeutic agent is chemically linked to an encapsulating wall ormatrix, the linkage or any spacer arm associated therewith mayadvantageously contain one or more labile groups which are cleavable torelease the agent. Representative cleavable groups include amide, imide,imine, ester, anhydride, acetal, carbamate, carbonate, carbonate esterand disulphide groups which are biodegradable in vivo, e.g. as a resultor hydrolytic and/or enzymatic action.

Representative and non-limiting examples of drugs useful in accordancewith this embodiment of the invention include antineoplastic agents suchas vincristine, vinblastine, vindesine, busulfan, chlorambucil,spiroplatin, cisplatin, carboplatin, methotrexate, adriamycin,mitomycin, bleomycin, cytosine arabinoside, arabinosyl adenine,mercaptopurine, mitotane, procarbazine, dactinomycin (antinomycin D),daunorubicin, doxorubicin hydrochloride, taxol, plicamycin,aminoglutethimide, estramustine, flutamide, leuprolide, megestrolacetate, tamoxifen, testolactone, trilostane, amsacrine (m-AMSA),asparaginase (L-asparaginase), etoposide, interferon a-2a and 2b, bloodproducts such as hematoporphyrins or derivatives of the foregoing;biological response modifiers such as muramylpeptides; antifungal agentssuch as ketoconazole, nystatin, griseofulvin, flucytosine, miconazole oramphotericin B; hormones or hormone analogues such as growth hormone,melanocyte stimulating hormone, estradiol, beclomethasone dipropionate,betamethasone, cortisone acetate, dexamethasone, flunisolide,hydrocortisone, methylprednisolone, paramethasone acetate, prednisolone,prednisone, triamcinolone or fludrocortisone acetate; vitamins such ascyanocobalamin or retinoids; enzymes such as alkaline phosphatase ormanganese superoxide dismutase; antiallergic agents such as amelexanox;anticoagulation agents such as warfarin, phenprocoumon or heparin;antithrombotic agents; circulatory drugs such as propranolol; metabolicpotentiators such as glutathione; antituberculars such asp-aminosalicylic acid, isoniazid, capreomycin sulfate, cyclosexine,ethambutol, ethionamide, pyrazinamide, rifampin or streptomycinsulphate; antivirals such as acyclovir, amantadine, azidothymidine,ribavirin or vidarabine; blood vessel dilating agents such as diltiazem,nifedipine, verapamil, erythritol tetranitrate, isosorbide dinitrate,nitroglycerin or pentaerythritol tetranitrate; antibiotics such asdapsone, chloramphenicol, neomycin, cefaclor, cefadroxil, cephalexin,cephradine, erythromycin, clindamycin, lincomycin, amoxicillin,ampicillin, bacampicillin, carbenicillin, dicloxacillin, cyclacillin,picloxacillin, hetacillin, methicillin, nafcillin, penicillin ortetracycline; antiinflammatories such as diflunisal, ibuprofen,indomethacin, meclefenamate, mefenamic acid, naproxen, phenylbutazone,piroxicam, tolmetin, aspirin or salicylates; antiprotozoans such aschloroquine, metronidazole, quinine or meglumine antimonate;antirheumatics such as penicillamine; narcotics such as paregoric;opiates such as codeine, morphine or opium; cardiac glycosides such asdeslaneside, digitoxin, digoxin, digitalin or digitalis; neuromuscularblockers such as atracurium mesylate, gallamine triethiodide,hexafluorenium bromide, metocurine iodide, pancuronium bromide,succinylcholine chloride, tubocurarine chloride or vecuronium bromide;sedatives such as amobarbital, amobarbital sodium, apropbarbital,butabarbital sodium, chloral hydrate, ethchlorvynol, ethinamate,flurazepam hydrochloride, glutethimide, methotrimeprazine hydrochloride,methyprylon, midazolam hydrochloride, paraldehyde, pentobarbital,secobarbital sodium, talbutal, temazepam or triazolam; localanaesthetics such as bupivacaine, chloroprocaine, etidocaine, lidocaine,mepivacaine, procaine or tetracaine; general anaesthetics such asdroperidol, etomidate, fentanyl citrate with droperidol, ketaminehydrochloride, methohexital sodium or thiopental and pharmaceuticallyacceptable salts (e.g. acid addition salts such as the hydrochloride orhydrobromide or base salts such as sodium, calcium or magnesium salts)or derivatives (e.g. acetates) thereof; and radiochemicals, e.g.comprising beta-emitters. Of particular importance are antithromboticagents such as vitamin K antagonists, heparin and agents withheparin-like activity such as antithrombin III, dalteparin andenoxaparin; blood platelet aggregation inhibitors such as ticlopidine,aspirin, dipyridamole, iloprost and abciximab; and thrombolytic enzymessuch as streptokinase and plasminogen activator. Other examples oftherapeutics include genetic material such as nucleic acids, RNA, andDNA of natural or synthetic origin, including recombinant RNA and DNA.DNA encoding certain proteins may be used in the treatment of manydifferent types of diseases. For example, tumour necrosis factor orinterleukin-2 may be provided to treat advanced cancers; thymidinekinase may be provided to treat ovarian cancer or brain tumors;interleukin-2 may be provided to treat neuroblastoma, malignant melanomaor kidney cancer; and interleukin-4 may be provided to treat cancer.

Contrast agent preparations in accordance with the invention may be usedas vehicles for contrast-enhancing moieties for imaging modalities otherthan ultrasound, for example X-ray, light imaging, magnetic resonanceand, more preferably, scintigraphic imaging agents. Controlled growth ofthe dispersed gas phase may be used to position such agents in areas ofinterest within the bodies of subjects, for example using ultrasoundirradiation of a target organ or tissue to induce the desired controlledgrowth and temporary retention of the agent, which may then be imagedusing the appropriate non-ultrasound imaging modality.

Contrast agent preparations in accordance with the invention may also beused as vehicles for therapeutically active substances which do notnecessarily require release from the preparation in order to exhibittheir therapeutic effect. Such preparations may, for example,incorporate radioactive atoms or ions such as beta-emitters whichexhibit a localised radiation-emitting effect following growth of thedispersed gas phase and temporary retention of the agent at a tergetsite. It will be appreciated that such agents should preferably bedesigned so that subsequent shrinkage and cessation of retention of thedispersed gas does not occur until the desired therapeutic radiationdosage has been administered.

Contrast agent preparations in accordance with the invention mayadditionally exhibit therapeutic properties in their own right. Thus,for example, the dispersed gas may be targeted to capillaries leading totumours and may act as cell toxic agents by blocking such capillaries.Thus it is possible by applying localised ultrasonic energy to obtain acontrolled and localised embolism; this may be of importance as such orin combination with other therapeutic measures. Concentrations ofdispersed gas in capillaries may also enhance absorption of ultrasonicenergy in hyperthermic therapy; this may be used in, for example,treatment of liver tumours. Irradiation with a relatively high energy(e.g. 5 W) focused ultrasound beam, e.g. at 1.5 MHz, may be appropriatein such applications.

It will be appreciated that the present invention extends topreparations comprising an aqueous medium having gas dispersed thereinand a composition comprising a diffusible component as generalcompositions of matter and to their use for non-imaging agent purposes.

The following non-limitative Examples serve to illustrate the invention.

EXAMPLE 1 Preparations

a) Perfluorobutane Gas Dispersion

Hydrogenated phosphatidylserine (100 mg) in a 2% solution of propyleneglycol in purified water (20 ml) was heated to 80ΕC for 5 minutes andthe resulting dispersion was allowed to cool to room temperatureovernight. 1 ml portions were transferred to 2 ml vials, the headspaceabove each portion was flushed with perfluorobutane gas, and the vialswere shaken for 45 seconds using an Espe CapMix7 mixer for dentalmaterials, yielding milky white microbubble dispersions with a volumemedian diameter of 5.0 μm, measured using a Coulter Counter (all CoulterCounter measurements were made at room temperature using an instrumentfitted with a 50 μm aperture and having a measuring range 1-30 μm;Isoton II was used as electrolyte).

b) Dispersion of Lyophilised Perfluorobutane Gas Dispersion

A sample of the milky white dispersion from Example 1(a) was washedthree times by centrifugation and removal of the infranatant, whereafteran equal volume of 10% sucrose solution was added. The resultingdispersion was lyophilised and then redispersed in distilled water,yielding a milky white microbubble dispersion with a volume mediandiameter of 3.5 μm, measured using a Coulter Counter.

c) 2-Methylbutane Emulsion

Hydrogenated phosphatidylserine (100 mg) in purified water (20 ml) washeated to 80ΕC for 5 minutes and the resulting dispersion was cooled to0ΕC overnight. 1 ml of the dispersion was transferred to a 2 ml vial, towhich was added 200 μl of 2-methylbutane (b.p. 28ΕC). The vial was thenshaken for 45 seconds using a CapMix7 to yield an emulsion of diffusiblecomponent which was stored at 0ΕC when not in use. The volume mediandiameter of the emulsion droplets was 1.9 μm, measured using a CoulterCounter.

d) Perfluoropentane Emulsion

The procedure of Example 1(c) was repeated except that the2-methylbutane was replaced by perfluoropentane (b.p. 29ΕC). Thethus-obtained emulsion of diffusible component was stored at 0ΕC whennot in use.

e) 2-Chloro-1,1,2-trifluoroethyl Difluoromethyl Ether Emulsion

The procedure of Example 1(c) was repeated except that the2-methylbutane was replaced by 2-chloro-1,1,2-trifluoroethyldifluoromethyl ether (b.p. 55-57ΕC). The thus-obtained emulsion ofdiffusible component was stored at 0ΕC when not in use.

f) 2-Bromo-2-chloro-1,1,1-trifluoroethane Emulsion

The procedure of Example 1(c) was repeated except that the2-methylbutane was replaced by 2-bromo-2-chloro-1,1,1-trifluoroethane(b.p. 49ΕC). The thus-obtained emulsion of diffusible component wasstored at 0ΕC when not in use.

g) 1-Chloro-2,2,2-trifluoroethyl Difluoromethyl Ether Emulsion

The procedure of Example 1(c) was repeated except that the2-methylbutane was replaced by 1-chloro-2,2,2-trifluoroethyldifluoromethyl ether (b.p. 49ΕC). The thus-obtained emulsion ofdiffusible component was stored at 0ΕC when not in use.

h) Dispersion of Gas-Containing Polymer/Human Serum Albumin Particles

Human serum albumin-coated gas-containing particles of polymer made fromethylidene bis(16-hydroxyhexadecanoate) and adipoyl chloride, preparedaccording to Example 3(a) of WO-A-9607434, (100 mg) were crushed in amortar and dispersed in 0.9% aqueous sodium chloride (10 ml) by shakingon a laboratory shaker for 24 hours.

i) Dispersion of Gas-Containing Polymer/Gelatin Particles

Gelatin-coated gas-containing particles of polymer made from ethylidenebis(16-hydroxyhexadecanoate) and adipoyl chloride, prepared according toExample 3(e) of WO-A-9607434, (100 mg) were crushed in a mortar anddispersed in 0.9% aqueous sodium chloride (10 ml) by shaking on alaboratory shaker for 24 hours.

j) 2-Methylbutane Emulsion

The procedure of Example 1(c) was repeated except that the emulsion wasdiluted 10 times prior to use and was stored in an ice bath when not inuse.

k) Perfluoropentane Emulsion

The procedure of Example 1(d) was repeated except that the emulsion wasdiluted 10 times prior to use and was stored in an ice bath when not inuse.

l) Perfluoropentane Emulsion

Hydrogenated phosphatidylserine (100 mg) in purified water (20 ml) washeated to 80ΕC for 5 minutes and the resulting dispersion was cooled to0ΕC overnight. 1 ml of the dispersion was transferred to a 2 ml vial, towhich was added 100 μl of perfluoro-n-pentane (b.p. 29ΕC). The vial wasthen shaken for 75 seconds using a CapMix7 to yield an emulsion ofdiffusible component which was stored at 0ΕC when not in use. The volumemedian diameter of the emulsion droplets was 2.9 am, measured using aCoulter Counter.

m) Perfluorobutane Emulsion

The procedure of Example 1(l) was repeated except that theperfluoropentane was replaced by perfluorobutane (b.p. −2ΕC). Thethus-obtained emulsion of diffusible component was stored at 0ΕC whennot in use.

n) Perfluoropentane Emulsion Prepared by Sonication

Hydrogenated phosphatidylserine (500 mg) in purified water (100 ml) washeated to 80° C. for 5 minutes and the resulting dispersion was allowedto cool to room temperature overnight. 10 ml of the dispersion weretransferred to a 30 ml vial, to which perfluoropentane (1 ml) was thenadded. Sonication of the resulting mixture for two minutes gave adispersion of diffusible component wherein the drops had a mean diameter<1 μm.

o) Perfluoropentane Emulsion

The procedure of Example 1(l) was repeated except that the volume ofperfluoropentane employed was reduced to 60 μl. The thus-obtainedemulsion of diffusible component was stored at 0° C. when not in use.

p) Perfluoropentane Emulsion

The procedure of Example 1(l) was repeated except that the volume ofperfluoropentane employed was reduced to 20 μl. The thus-obtainedemulsion of diffusible component was stored at 0° C. when not in use.

q) Perfluoropentane:perfluoro-4-methylpent-2-ene (1:1) Emulsion

The procedure of Example 1(l) was repeated except that theperfluoropentane was replaced by a mixture of 50 μl of perfluoropentane(b.p. 29ΕC) and 50 μl of perfluoro-4-methylpent-2-ene (b.p. 49ΕC). Thethus-obtained emulsion of diffusible components was stored at 0ΕC whennot in use. The volume median diameter of the emulsion droplets was 2.8μm, measured using a Coulter Counter.

r) Perfluoropentane:1H,1H,2H-heptafluoropent-1-ene (1:1) Emulsion

The procedure of Example 1(l) was repeated except that theperfluoropentane was replaced by a mixture of 50 μl of perfluoropentane(b.p. 29ΕC) and 50 μl of 1H,1H,2H-heptafluoropent-1-ene (b.p. 30-31ΕC).The thus-obtained emulsion of diffusible components was stored at 0ΕCwhen not in use.

s) Perfluoropentane Emulsion Stabilised byDistearoylphosphatidylcholine:Distearoylphosphatidyl-Serine (1:1)

The procedure of Example 1(l) was repeated except that the hydrogenatedphosphatidylserine was replaced by a mixture ofdistearoylphosphatidylcholine (50 mg) and distearoylphosphatidylserine,sodium salt (50 mg). The thus-obtained emulsion of diffusible componentwas stored at 0ΕC when not in use.

t) Perfluoropentane Emulsion Stabilised byDistearoylphosphatidylcholine:Distearoylphosphatidyl-Serine (3:1)

The procedure of Example 1(l) was repeated except that the hydrogenatedphosphatidylserine was replaced by a mixture ofdistearoylphosphatidylcholine (75 mg) and distearoylphosphatidylserine,sodium salt (25 mg). The thus-obtained emulsion of diffusible componentwas stored at 0ΕC when not in use.

u) Perfluoropentane Emulsion Stabilised byDistearoylphosphatidylcholine:Distearoylphosphatidyl-Glycerol (3:1)

The procedure of Example 1(l) was repeated except that the hydrogenatedphosphatidylserine was replaced by a mixture ofdistearoylphosphatidylcholine (75 mg) anddistearoylphosphatidylglycerol, sodium salt (25 mg). The thus-obtainedemulsion of diffusible component was stored at 0ΕC when not in use.

v) Perfluoropentane Emulsion Stabilised by HydrogenatedPhosphatidylcholine:Hydrogenated Phosphatidylserine (11:1)

The procedure of Example 1(l) was repeated except that the hydrogenatedphosphatidylserine was replaced by 100 mg of a mixture of hydrogenatedphosphatidylcholine and hydrogenated phosphatidylserine (11:1). Thethus-obtained emulsion of diffusible component was stored at 0ΕC whennot in use.

w) Perfluoro-4-methylpent-2-ene Emulsion Stabilised byDistearoylphosphatidylcholine:Distearoylphosphatidyl-Serine (3:1)

The procedure of Example 1(l) was repeated except that the hydrogenatedphosphatidylserine was replaced by a mixture ofdistearoylphosphatidylcholine (75 mg) and distearoylphosphatidylserine,sodium salt (25 mg) and the perfluoropentane was replaced byperfluoro-4-methylpent-2-ene. The thus-obtained emulsion of diffusiblecomponent was stored at 0ΕC when not in use.

x) Perfluoropentane:perfluoro-4-methylpent-2-ene (1:1) EmulsionStabilised by Distearoylphosphatidylcholine:Distearoylphosphatidylserine(3:1)

The procedure of Example 1(w) was repeated except that theperfluoro-4-methylpent-2-ene was replaced by a mixture of 50 μl ofperfluoropentane and 50 μl of perfluoro-4-methylpent-2-ene. Thethus-obtained emulsion of diffusible components was stored at 0ΕC whennot in use.

v) Perfluoropentane:perfluoro-4-methylpent-2-ene (1:1) EmulsionStabilised byDistearoylphosphatidylcholine:Distearoylphosphatidylglycerol (3:1)

The procedure of Example 1(x) was repeated except that thedistearoylphosphatidylserine, sodium salt was replaced bydistearoylphosphatidylglycerol, sodium salt. The thus-obtained emulsionof diffusible components was stored at 0ΕC when not in use.

z) Perfluorodecalin:Perfluorobutane Emulsion

Hydrogenated phosphatidylserine (100 mg) in aqueous glycerol(5.11%)/propylene glycol (1.5%) (20 ml) was heated to 80ΕC for 5 minutesand the resulting dispersion was cooled to 0ΕC overnight. 1 ml of thedispersion was transferred to a 2 ml vial, to which was added 100Φl ofperfluorodecalin (b.p. 141-143ΕC) saturated with perfluorobutane (b.p.−2ΕC). The vial was then shaken for 60 seconds using a CapMix7 to yieldan emulsion of diffusible component which was stored at 0ΕC when not inuse.

aa) Perfluorodecalin:Perfluoropropane Emulsion

The procedure of Example 1(z) was repeated except that theperfluorodecalin saturated with perfluorobutane was replaced byperfluorodecalin saturated with perfluoropropane (b.p. −39ΕC). Thethus-obtained emulsion of diffusible component was stored at 0ΕC whennot in use.

ab) Perfluorodecalin:Sulphur Hexafluoride Emulsion

The procedure of Example 1(z) was repeated except that theperfluorodecalin saturated with perfluorobutane was replaced byperfluorodecalin saturated with sulphur hexafluoride (b.p. −64ΕC). Thethus-obtained emulsion of diffusible component was stored at 0ΕC whennot in use.

ac) Perfluoropentane Emulsion Stabilised with Fluorad FC-170C

1 ml of a dispersion of Fluorad FC-170C (200 mg) in purified water (20ml) was transferred to a 2 ml vial, to which was added 100 Φl ofperfluoro-n-pentane. The vial was then shaken for 75 seconds using aCapMix7 to yield an emulsion of diffusible component which was stored at0ΕC when not in use.

ad) Perfluoropentane Emulsion Stabilised with Pluronic F68:FluoradFC-170C

100 Φl of a 10% Pluronic F68 solution was added to 200 Φl of 1% FluoradFC170C and 700 Φl purified water. The resulting mixture was transferredto a 2 ml vial, to which was added 100 Φl of perfluoro-n-pentane. Thevial was then shaken for 75 seconds using a CapMix7 to yield an emulsionof diffusible component which was stored at 0ΕC when not in use. Asample of this emulsion was transferred to a screw-topped plastic vial(2.8 ml) which was then sonicated in a water bath for 2 minutes (pulsesonication: 1 per second). The volume median diameter of the sonicatedemulsion droplets was 0.99Φm, measured using a Coulter Counter.

ae) Perfluoropentane Emulsion Stabilised with Pluronic F68:FluoradFC-170C and Prepared by Homogenisation

1 ml of a 10% Pluronic F68 solution was added to 2 ml of 1% FluoradFC170C and 7 ml purified water, whereafter 1 ml of perfluoro-n-pentanewas added to the resulting mixture. The thus-obtained dispersion wasthen homogenised by rotor/stator homogenisation for 2 minutes at 23000rpm. The resulting emulsion was transferred to a screw-topped plasticvial (10 ml) and sonicated in a water bath for 2 minutes (pulsesonication: 1 per second).

af) Perfluoropentane Emulsion

Hydrogenated phosphatidylserine (250 mg) in purified water (100 ml) washeated to 80° C. for 5 minutes and the resulting dispersion was cooledto 0° C. overnight. 1 ml of the dispersion was transferred to a 2 mlvial, to which was added 100 μl of perfluoropentane. The vial was shakenfor 75 seconds using a CapMix⁷ to yield an emulsion of diffusiblecomponent which was stored at 0° C. when not in use.

ag) Dispersion of Lyophilised Perfluorobutane Gas Dispersion

A sample of the milky white dispersion from Example 1(a) was washedthree times by centrifugation and removal of the infranatant, whereafteran equal volume of 10% sucrose solution was added. The resultingdispersion was lyophilised and then redispersed in distilled water,yielding a milky white microbubble dispersion with a volume meandiameter of 2.6 Φm, measured using a Coulter Counter.

ah) Perfluoropropane Gas Dispersion

The procedure of Example 1(a) was repeated except that theperfluorobutane gas was replaced by perfluoropropane gas.

The resulting milky white microbubble dispersion had a volume mediandiameter of 2.6 Φm, measured using a Coulter Counter.

ai) Perfluoropentane Emulsion

Hydrogenated phosphatidylserine (100 mg) in purified water (100 ml) washeated to 80° C. for 5 minutes and the resulting dispersion was cooledto 0° C. overnight. 1 ml of the dispersion was transferred to a 2 mlvial, to which was added 100 μl of perfluoropentane. The vial was shakenfor 75 seconds using a CapMix⁷ to yield an emulsion of diffusiblecomponent which was stored at 0° C. when not in use.

aj) Perfluoropentane Emulsion Stabilised with Brij 58:Fluorad FC-170C,Prepared by Shaking

Brij 58 (400 mg) was added to a solution of 0.1% Fluorad FC-170C (10 ml)and stirred at room temperature for one hour. 1 ml of the resultingsolution was transferred to a 2 ml vial, to which was addedperfluoropentane (100 μl). The vial was then shaken for 75 seconds usinga CapMix⁷ to yield an emulsion of diffusible component which was storedat 0° C. when not in use.

ak) Perfluoropentane Emulsion Stabilised with Brij 58:Fluorad FC-170C,Prepared by Sonication

Brij58 (400 mg) was added to a solution of 0.1% Fluorad FC-170C (10 ml)and stirred at room temperature for one hour. Perfluoropentane (1 ml)was then added and the resulting mixture was sonicated for 2 minutes toyield an emulsion of small drops of the diffusible component. Thisemulsion was stored at 0° C. when not in use.

al) Perfluoro-4-methylpent-2-ene Emulsion

The procedure of Example 1(l) was repeated except that theperfluoropentane was replaced by perfluoro-4-methylpent-2-ene (b.p. 49°C.). The thus-obtained emulsion of diffusible component was stored at 0°C. when not in use.

am) 1H,1H,2H-Heptafluoropent-1-ene Emulsion

The procedure of Example 1(l) was repeated except that theperfluoropentane was replaced by 1H,1H,2H-heptafluoropent-1-ene (b.p.30-31° C.). The thus-obtained emulsion of diffusible component wasstored at 0° C. when not in use.

an) Perfluorocyclopentene Emulsion

The procedure of Example 1(l) was repeated except that theperfluoropentane was replaced by perfluorocyclopentene (b.p. 27° C.).The thus-obtained emulsion of diffusible component was stored at 0° C.when not in use.

ao) Perfluorodimethylcyclobutane Emulsion

The procedure of Example 1(l) was repeated except that theperfluoropentane was replaced by perfluorodimethyl-cyclobutane (mixtureof 1,2- and 1,3-isomers, b.p. 45° C.). The thus-obtained emulsion ofdiffusible component was stored at 0° C. when not in use.

ap) Emulsion of an Azeotropic Perfluorohexane:n-Pentane Mixture

4.71 g (0.014 mol) perfluoro-n-hexane (boiling point 59ΕC)(FluorochemLtd.) and 0.89 g (0.012 mol) n-pentane (boiling point 36ΕC) (Fluka AG)were mixed in a vial to give an azeotropic mixture shown to boil readilyat 35ΕC. In another vial, hydrogenated phosphatidylserine (100 mg) inpurified water (20 ml) was heated to 80ΕC for 5 minutes and theresulting dispersion was cooled to room temperature. 1 ml of thephospholipid dispersion was transferred to a 2 ml vial to which wasadded 100 μl of the azeotropic mixture. The vial was then shaken for 45seconds using a CapMix⁷ to yield an emulsion of diffusible componentwhich was stored at room temperature when not in use.

ag) Perfluorodimethylcyclobutane Emulsion

The procedure of Example 1(l) was repeated except that theperfluoropentane was replaced by perfluorodimethyl-cyclobutane (97%1,1-isomer, balance being 1,2- and 1,3-isomers). The thus-obtainedemulsion of diffusible component was stored at 0° C. when not in use.

ar) Perfluorohexane Emulsion

The procedure of Example 1(l) was repeated except that theperfluoropentane was replaced by perfluorohexane (b.p. 57ΕC). Thethus-obtained emulsion of diffusible component was stored at 0ΕC whennot in use.

as) Perfluorodimethylcyclobutane Emulsion Stabilised with FluorinatedSurfactant

The procedure of Example 1(aq) is repeated except that the hydrogenatedphosphatidylserine is replaced by either perfluorinateddistearoylphosphatidylcholine (5 mg/ml) or a mixture of perfluorinateddistearoylphosphatidylcholine and hydrogenated phosphatidylserine (3:1,total lipid concentration 5 mg/ml). The thus-obtained emulsions ofdiffusible component was stored at 0ΕC when not in use.

at) 2,2,3,3,3-Pentafluoropropyl Methyl Ether Emulsion

The procedure of Example 1(l) was repeated except that theperfluoropentane was replaced by 2,2,3,3,3-pentafluoropropyl methylether (b.p. 46ΕC). The thus-obtained emulsion of diffusible componentwas stored at 0ΕC when not in use.

au) 2H,3H-Decafluoropentane Emulsion

The procedure of Example 1(l) was repeated except that theperfluoropentane was replaced by 2H,3H-decafluoropentane (b.p.54ΕC). Thethus-obtained emulsion of diffusible component was stored at 0ΕC whennot in use.

av) Perfluorodimethylcyclobutane Emulsion Stabilised byLysophosphatidylcholine

The procedure of Example 1(aq) was repeated except that the hydrogenatedphosphatidylserine was replaced by lysophosphatidylcholine. Thethus-obtained emulsion of diffusible component was stored at 0ΕC whennot in use.

aw) Perfluorodimethylcyclobutane Emulsion Stabilised by HydrogenatedPhosphatidylserine:Lysophosphatidyl-Choline (1:1)

The procedure of Example 1(aq) was repeated except that the hydrogenatedphosphatidylserine was replaced by a mixture of hydrogenatedphosphatidylserine and lysophosphatidylcholine (1:1). The thus-obtainedemulsion of diffusible component was stored at 0ΕC when not in use.

ax) Perfluorodimethylcyclobutane Emulsion Stabilised by a PolyethyleneGlycol 10,000-Based Surfactant

The procedure of Example 1(aq) was repeated except that the hydrogenatedphosphatidylserine dispersion was replaced by a solution of□-(16-hexadecanoyloxy-hexadecanoyl)-□-methoxypolyethylene glycol 10,000in water (10 mg/ml). The thus-obtained emulsion of diffusible componentwas stored at 0ΕC when not in use.

av) Perfluorodimethylcyclobutane Emulsion Stabilised by a PolyethyleneGlycol 10,000-Based Surfactant

The procedure of Example 1(aq) was repeated except that the hydrogenatedphosphatidylserine dispersion was replaced by a solution of□-(16-hexadecanoyloxy-hexadecanoyl)-□-methoxypolyethylene glycol 10,000in water (20 mg/ml). The thus-obtained emulsion of diffusible componentwas stored at 0ΕC when not in use.

az) Perfluorobutane-Filled Microbubbles Encapsulated byPhosphatidylserine and RGDC-Mal-Polyethylene Glycol2000-Distearoylphosphatidylethanolamine

To a mixture of phosphatidylserine (4.5 mg) and Mal-polyethylene glycol2000-distearoylphosphatidyl-ethanolamine (0.5 mg) in a vial was added asolution of 1.4% propylene glycol/2.4% glycerol in water (1 ml). Thedispersion was heated to 80ΕC for 5 minutes, cooled to room temperatureand then flushed with perfluorobutane gas. The vial was shaken for 45seconds using a CapMix7 and then placed on a roller table. Aftercentrifugation the infranatant was exchanged with a solution of RGDC insodium phosphate buffer having a pH of 7.5, after which the vial wasplaced on the roller table for several hours.

ba) Perfluorobutane-Filled Microbubbles Encapsulated byPhosphatidylserine and Dipalmitoylphosphatidyl-Ethanolamine-PolyethyleneGlycol 2000

To a vial containing phosphatidylserine anddipalmitoylphosphatidylethanolamine-polyethylene glycol 2000 (ratio10:1) is added a solution of 2% propylene glycol in water to give alipid concentration of 5 mg/ml. The dispersion is heated to 80ΕC for 5minutes and then cooled to room temperature, whereafter the headspace isflushed with perfluorobutane gas. The vial is shaken for 45 secondsusing a CapMix7 and is then placed on a roller table. After washing bycentrifugation and removal of infranatant, an equal volume of watercontaining 10% sucrose is added. The resulting dispersion is lyophilisedand then redispersed by adding water, yielding a milky white microbubbledispersion.

bb) Perfluorobutane-Filled Microbubbles Encapsulated byPhosphatidylserine and Distearoylphosphatidyl-Ethanolamine-PolyethyleneGlycol 5000

To a vial containing phosphatidylserine anddistearoylphosphatidylethanolamine-polyethylene glycol 5000 (ratio 10:1)is added a solution of 2% propylene glycol in water to give a lipidconcentration of 5 mg/ml. The dispersion is heated to 80ΕC for 5 minutesand then cooled to room temperature, whereafter the headspace is flushedwith perfluorobutane gas. The vial is shaken for 45 seconds using aCapMix7 and is then placed on a roller table. After washing bycentrifugation and removal of infranatant, an equal volume of watercontaining 10% polyethylene glycol is added. The resulting dispersion islyophilised and then redispersed, yielding a milky white microbubbledispersion.

bc) Perfluorobutane-Filled Microbubbles Encapsulated byPhosphatidylserine and Dipalmitoylphosphatidyl-Ethanolamine-PolyethyleneGlycol 2000

To a vial containing phosphatidylserine anddipalmitoylphosphatidylethanolamine-polyethylene glycol 2000 (ratio10:1) is added a solution of 2% propylene glycol in water to give alipid concentration of 5 mg/ml. The dispersion is heated to 80ΕC for 5minutes and then cooled to room temperature, whereafter the headspace isflushed with perfluorobutane gas. The vial is shaken for 45 secondsusing a CapMix7 and is then placed on a roller table. After washing bycentrifugation and removal of infranatant, an equal volume of water isadded, yielding a milky white microbubble dispersion.

bd) Perfluorobutane-Filled Microbubbles Encapsulated byPhosphatidylserine and Distearoylphosphatidyl-Ethanolamine-PolyethyleneGlycol 5000

To a vial containing phosphatidylserine anddistearoylphosphatidylethanolamine-polyethylene glycol 5000 (ratio 10:1)is added a solution of 2% propylene glycol in water to give a lipidconcentration of 5 mg/ml. The dispersion is heated to 80ΕC for 5 minutesand then cooled to room temperature, whereafter the headspace is flushedwith perfluorobutane gas. The vial is shaken for 45 seconds using aCapMix7 and is then is placed on a roller table. After washing bycentrifugation and removal of infranatant, an equal volume of water isadded, yielding a milky white microbubble dispersion.

be) Perfluorodimethylcyclobutane Emulsion Stabilised byPhosphatidylserine and Dipalmitoylphosphatidyl-Ethanolamine-PolyethyleneGlycol 2000

The procedure of Example 1(aq) is repeated except that the hydrogenatedphosphatidylserine is replaced by a mixture of hydrogenatedphosphatidylserine and dipalmitoylphosphatidylethanolamine-polyethyleneglycol 2000 (ratio 10:1). The thus-obtained emulsion of diffusiblecomponent is stored at 0ΕC when not in use.

bf) Perfluorodimethylcyclobutane Emulsion Stabilised byPhosphatidylserine and Distearoylphosphatidyl-Ethanolamine-PolyethyleneGlycol 5000

The procedure of Example 1(aq) is repeated except that the hydrogenatedphosphatidylserine is replaced by a mixture of hydrogenatedphosphatidylserine and distearoylphosphatidylethanolamine-polyethyleneglycol 5000 (ratio 10:1). The thus-obtained emulsion of diffusiblecomponent is stored at 0ΕC when not in use.

bk) Lyophilised Perfluorobutane-Filled Microbubbles Redispersed in anEmulsion

A sample of the milky white dispersion prepared as described in Example1(bp) was washed three times by centrifugation and removal of theinfranatant, whereafter an equal volume of 10% sucrose solution wasadded. The resulting dispersion was lyophilised and then redispersed inan emulsion prepared as described in Example 1(aq) just prior to use.

bh) Avidinylated Perfluorodimethylcyclobutane Emulsion Droplets

Distearoylphosphatidylserine (4.5 mg) andbiotin-dipalmitoylphosphatidylethanolamine (0.5 mg) were weighed into aclean vial and 1.0 ml of a solution of 2% propylene glycol was added.Following heating to 80° C. the mixture was cooled to room temperature.100 Φl of perfluorodimethylcyclobutane were added and the vial wasshaken for 75 seconds using a CapMix7 to yield an emulsion of diffusiblecomponent. A diluted sample of the emulsion (100 μl emulsion in 1 mlwater) was incubated with excess avidin and placed on a roller table.The diluted emulsion was then washed extensively with water andconcentrated by centrifuging.

bi) 1H-Tridecafluorohexane Emulsion

The procedure of Example 1(l) was repeated except that theperfluoropentane was replaced by 1H-tridecafluorohexane (b.p. 71ΕC). Thethus-obtained emulsion of diffusible component was stored at 0ΕC whennot in use.

bi) Perfluoroheptane Emulsion

The procedure of Example 1(l) was repeated except that theperfluoropentane was replaced by perfluoroheptane (b.p. 80-85ΕC). Thethus-obtained emulsion of diffusible component was stored at 0ΕC whennot in use.

bk) Perfluorodimethylcyclobutane emulsion with phosphatidylserine andfluorescent streptavidin Distearoylphosphatidylserine (4.5 mg) andbiotin-dipalmitoylphosphatidylethanolamine (0.5 mg) are weighed into aclean vial and 1.0 ml of a solution of 2% propylene glycol is added.Following heating to 80° C. the mixture is cooled to room temperature.100 Φl of perfluorodimethylcyclobutane are added and the vial is shakenfor 75 seconds using a CapMix7 mixer to yield an emulsion of diffusiblecomponent. A diluted sample of the emulsion (100Φl emulsion in 1 mlwater) is incubated with excess fluorescent streptavidin in phosphatebuffer and placed on a roller table. The diluted emulsion is then washedextensively with water and concentrated by centrifuging.

bl) Dispersion of Lyophilised Perfluorobutane Gas Dispersion

A sample of the milky white dispersion prepared as described in Example1(a) was washed three times by centrifugation and removal of theinfranatant, whereafter an equal volume of 10% sucrose solution wasadded. The resulting dispersion was lyophilised and then redispersed indistilled water just prior to use.

bm) Perfluorodimethylcyclobutane Emulsion Stabilised by SterilisedPhosphatidylserine

The procedure of Example 1(aq) was repeated except that the hydrogenatedphosphatidylserine was replaced by a sterilised solution of hydrogenatedphosphatidylserine. The thus-obtained emulsion of diffusible componentwas stored at 0ΕC when not in use.

bn) Perfluoropropane Gas Dispersion

The procedure of Example 1(a) was repeated except that theperfluorobutane gas was replaced by perfluoropropane gas.

bo) Dispersed Echovist

Echovist granulate (Schering AG) (0.25 g) was dispersed in an emulsion(1.15 ml) prepared as described in Example 1(aq).

bp) Perfluorobutane Gas Dispersion

Hydrogenated phosphatidylserine (500 mg) was added to a solution of 1.5%propylene glycol/5.11% glycerol in water (100 ml) and heated to 80ΕC for5 minutes, whereafter the resulting dispersion was allowed to cool toambient temperature. 1 ml portions were transferred to 2 ml vials, theheadspace above each portion was flushed with perfluorobutane gas, andthe vials were shaken for 45 seconds using a CapMix7, whereafter thevials were placed on a roller table.

ba) Preparation of Biotinylated Perfluorobutane Microbubbles

Distearoylphosphatidylserine (4.5 mg) andbiotin-dipalmitoylphosphatidylethanolamine (0.5 mg) were weighed into aclean vial and 1.0 ml of a solution of 1.4% propylene glycol/2.4%glycerol was added. Following heating to 78° C. the mixture was cooledto room temperature and the head space was flushed with perfluorobutanegas. The vial was shaken for 45 seconds using a CapMix7 mixer and wasthen placed on a roller table for 16 hours. The resulting microbubbleswere washed extensively with deionised water.

br) Aerogels

To “spatula edge” pyrolysed resorcinol-formaldehyde aerogel particles(provided by Dr. Pekala, Lawrence Livermore National Laboratory) wereadded 300 μl water, a droplet of pH9 buffer and 5-10 droplets of 1%Pluronic F68. The aerogel particles sedimented quickly, but did notaggregate.

bs) Small Bubbles

A rubber tube, 8 mm inner diameter and approximately 20 cm long, wasplaced vertically in a stand, capped at the bottom end and filled with amicrobubble dispersion made according to Example 1(a) (except that anYstral7 rotor stator homogeniser was used to make the microbubbledispersion). After two hours, a syringe connected to a canula wasinserted into the rubber tube close to the bottom, and a 1 ml fractionof the size-fractionated microbubble dispersion was collected. Coultercounter analysis revealed the thus-obtained microbubble dispersion tohave a median diameter of 1.2 μm.

bt) Perfluorobutane Gas Dispersion Stabilised by 5% Albumin:5% Dextrose(1:3)

20% human serum was diluted to 5% with purified water. A 5 ml sample ofthe diluted albumin was further diluted with 5% glucose (15 ml) and theresulting mixture was transferred to a vial. The head space was flushedwith perfluorobutane gas and the vial was sonicated for 80 seconds,giving a milky white microbubble dispersion.

bu) Dispersion of Buckminsterfullerene C₆₀

Buckminsterfullerene C₆₀ was added to 2.5% human serum albumin (1 ml) ina 2 ml vial which was shaken for 75 seconds using a CapMix7.

bv) Sulphur Hexafluoride Gas Dispersion

Distearoylphosphatidylcholine:dipalmitoylphosphatidylglycerol (10:1)stabilised microbubbles were made as described in Example 5 ofWO-A-9409829. Thus 50 mg distearoylphosphatidylcholine, 5 mgdipalmitoylphosphatidylglycerol and 2.2 g polyethylene glycol 4000 weredissolved in 22 ml t-butanol at 60° C., and the solution was rapidlycooled to −77° C. and lyophilised overnight. 100 mg of the resultingpowder were placed in a vial, and the head space was evacuated and thenfilled with sulphur hexafluoride. 1 ml purified water was added justbefore use, giving a microbubble dispersion.

bw) 2-Methylbutane Emulsion

Hydrogenated phosphatidylserine (100 mg) in purified water (20 ml) washeated to 80° C. for 5 minutes and the resulting dispersion was cooledin refrigerator overnight. 1 ml of the dispersion was transferred to a 2ml vial, to which was added 100 μl of 2-methylbutane. The vial wasshaken for 75 seconds using a CapMix7 to yield an emulsion of diffusiblecomponent which was stored at 0° C. when not in use.

bx) Lyophilised Perfluorobutane Gas Dispersion in Aqueous SodiumBicarbonate

A sample of the milky white dispersion from Example 1(a) was washedthree times by centrifugation and removal of the infranatant, whereafteran equal volume of 10% sucrose solution was added. The resultingdispersion was lyophilised and then redispersed in 0.1M sodiumbicarbonate solution.

by) Perfluorobutane Gas Dispersion

A perfluorobutane gas dispersion was prepared as Example 1(a). Thedispersion was washed three times with purified water by centrifugationand removal of the infranant, yielding a milky white microbubbledispersion.

bz) Perfluorobutane Gas Dispersion with Iron Oxide Particles

To 1 ml of a perfluorobutane gas dispersion prepared as in Example 1(by)was added 1 ml purified water. The pH was raised to 11.2 with ammoniumhydroxide and the dispersion was heated for 5 minutes at 60° C. Uncoatediron oxide particles (0.3 ml, 4.8 mg Fe/ml) were added and thedispersion was allowed to stand for 5 minutes. The pH was lowered to 5.9with hydrochloric acid, yielding a brown dispersion which after a whilegave a top layer with brown particles, a clear non-coloured infranantand no precipitate.

ca) Perfluorobutane Gas Dispersion with Iron Oxide Particles

To 1 ml of a perfluorobutane gas dispersion prepared as in Example 1(by)was added 0.3 ml uncoated iron oxide particles (4.8 mg Fe/ml) at pH 7,yielding a brown dispersion which on standing gave a top layer withbrown microbubbles, a clear infranant and no precipitate.

cb) [Comparative]

To 1 ml of a solution of hydrogenated phosphatidylserine in purifiedwater (5 mg/ml) was added 0.3 ml uncoated iron oxide particles (4.8 mgFe/ml) yielding a brown dispersion which after standing gave a brownprecipitate.

cc) Perfluorobutane Gas Dispersion with Iron Oxide Particles Coated withOleic Acid

1.3 mmol FeCl₂ 4H₂O (0.259 g) and 2.6 mmol FeCl₃6H₂O (0.703 g) weredissolved in 10 ml purified water and 1.5 ml ammonium hydroxide wereadded. The resulting iron oxide particles were washed five times withpurified water (25 ml). Diluted ammonium hydroxide was added to theparticles and the suspension was heated to 80° C. Oleic acid (0.15 g)was added, and the dispersion was allowed to stand for 5 minutes atambient temprature. Purified water (10 ml) was added and the pH waslowered to 5.4 with hydrochloric acid. The dispersion was sonicated for15 minutes, whereafter the infranant was removed and the particles weresuspended in 2-methylbutane (5 ml), yielding a fine black dispersion.

25 mg distearoylphosphatidylcholine and 2.5 mgdimyristoylphosphatidylglycerol were dissolved in 11 ml t-butanol at 60°C. and 0.1 ml iron oxide particles from above was added, together with1.1 g polyethylene glycol 4000. The dispersion was heated for 10 minutesat 60° C., rapidly cooled to −77° C. and lyophilised. 100 mg of thelyophilisate were introduced into a 2 ml vial, which was then evacuatedand flushed twice with perfluorobutane gas. The lyophilisate was thendispersed in 1 ml purified water and washed twice with purified water bycentrifugation with removal of the infranant and the precipitate. Afterstanding the resulting dispersion had a light grey and floating toplayer.

EXAMPLE 2 In Vitro Characterisation of Microbubble Growth byMicroscopy/Visual Observation

a) One drop of the perfluorobutane gas dispersion from Example 1(a) atca. 4ΕC was diluted with one drop of air-supersaturated purified waterat ca. 4ΕC on a microscope object glass cooled to ca. 4ΕC andinvestigated at 400× magnification. The microbubbles were observed tovary in size from 2 to 5 μm. The temperature was then raised to ca.40ΕC, whereupon a significant increase in the size of the microbubbleswas observed, the larger microbubbles growing most in size. The numberof microbubbles was significantly reduced after about 5 minutes.

b) [comparative] One drop of the 2-methylbutane emulsion from Example1(c), cooled in an ice bath to ca. 0ΕC, was placed on a microscopeobject glass cooled to ca. 0ΕC and investigated at 400× magnification.The oil phase droplets of the emulsion were observed to vary in sizefrom 2 to 6 μm. The temperature was then raised to ca. 40ΕC. Nomicrobubble formation was observed.

c) A sample of the perfluorobutane gas dispersion from Example 1(a) (0.5ml) was diluted with purified water (50 ml) and cooled to 0ΕC. A portionof this diluted disperion (1 ml) was mixed with a portion of the2-methylbutane emulsion from Example 1(c) (100 μl). One drop of theresulting mixture was placed on a microscope object glass maintained at0ΕC by means of a heating/cooling stage and covered with a cover glass,also at 0ΕC. The temperature of the object glass was gradually increasedto 40ΕC using the heating/cooling stage. Rapid and substantialmicrobubble growth was observed by microscopy and was confirmed by sizeand distribution measurements made using a Malvern Mastersizer.

d) [comparative] A sample of the perfluorobutane gas dispersion fromExample 1(a) (0.5 ml) was diluted with purified water (50 ml) and cooledin an ice bath to 0ΕC. A portion of this diluted disperion (1 ml) wasmixed with 100 μl of a 5 mg/ml dispersion of hydrogenatedphosphatidylserine in purified water, also at 0ΕC. One drop of theresulting mixture was placed on a microscope object glass cooled to 0ΕCand investigated at 400× magnification. The microbubbles were observedto vary in size from 2 to 5 μm. The temperature was then raised to ca.40ΕC, whereupon a significant increase in the size of the microbubbleswas observed, although the increase was less heavy and less rapid thenthat observed in Example 2(c).

e) A sample of the perfluorobutane gas dispersion from Example 1(a) wasdiluted with purified water (1:1) and cooled to 0ΕC. A drop of the2-chloro-1,1,2-trifluoroethyl difluoromethyl ether emulsion from Example1(e) was added to the diluted microbubble dispersion on a microscopeobject glass maintained at 0ΕC by means of a heating/cooling stage andcovered with a cover glass, also at 0ΕC. The temperature of the objectglass was gradually increased to 40ΕC using the heating/cooling stage.Rapid and substantial microbubble growth was observed by microscopy.

f) A sample of the perfluorobutane gas dispersion from Example 1(a) wasdiluted with purified water (1:1) and cooled to 0ΕC. A drop of the2-bromo-2-chloro-1,1,1-trifluoroethane emulsion from Example 1(f) wasadded to the diluted microbubble dispersion on a microscope object glassmaintained at 0ΕC by means of a heating/cooling stage and covered with acover glass, also at 0ΕC. The temperature of the object glass wasgradually increased to 40ΕC using the heating/cooling stage.

Rapid and substantial microbubble growth was observed by microscopy.

g) A sample of the perfluorobutane gas dispersion from Example 1(a) wasdiluted with purified water (1:1) and cooled to 0ΕC. A drop of the1-chloro-2,2,2-trifluoroethyl difluoromethyl ether emulsion from Example1(g) was added to the diluted microbubble dispersion on a microscopeobject glass maintained at 0ΕC by means of a heating/cooling stage andcovered with a cover glass, also at 0ΕC. The temperature of the objectglass was gradually increased to 40ΕC using the heating/cooling stage.Rapid and substantial microbubble growth was observed by microscopy.

h) One drop of the dispersion of polymer/human serum albuminmicroparticles from Example 1(h) and one drop of the perfluoropentaneemulsion from Example 1(k) were placed on a microscope object glasswarmed to 50ΕC and investigated at 400× magnification. Significantgrowth of microbubbles was observed as the drops mixed.

i) One drop of the dispersion of polymer/human serum albuminmicroparticles from Example 1(h) and one drop of the 2-methylbutaneemulsion from Example 1(j) were placed on a microscope object glasswarmed to 40ΕC and investigated at 400× magnification. Significant,rapid and heavy growth of microbubbles was observed as the drops mixed.

j) One drop of the dispersion of polymer/gelatin microparticles fromExample 1(i) and one drop of the perfluoropentane emulsion from Example1(k) were placed on a microscope object glass warmed to 50ΕC andinvestigated at 400× magnification. Significant growth of microbubbleswas observed as the drops mixed.

k) One drop of the dispersion of polymer/gelatin microparticles fromExample 1(i) and one drop of the 2-methylbutane emulsion from Example1(j) were placed on a microscope object glass warmed to 40ΕC andinvestigated at 400× magnification. Significant, rapid and heavy growthof microbubbles was observed as the drops mixed.

l) [comparative] One drop of the perfluoropentane emulsion from Example1(k) was placed on a microscope object glass warmed to 50ΕC andinvestigated at 400× magnification. No microbubble formation wasobserved.

m) [comparative] One drop of the 2-methylbutane emulsion from Example1(j) was placed on a microscope object glass warmed to 40ΕC andinvestigated at 400× magnification. No microbubble formation wasobserved.

n) [comparative] One drop of the dispersion of polymer/human serumalbumin microparticles from Example 1(h) is placed on a microscopeobject glass warmed to 40ΕC and investigated at 400× magnification. Nosignificant change is seen.

o) [comparative] One drop of the dispersion of polymer/gelatinmicroparticles from Example 1(i) is placed on a microscope object glasswarmed to 50ΕC and investigated at 400× magnification. No significantchange is seen.

p) One drop of a dispersion of human serum albumin-stabilised airmicrobubbles prepared as described in US-A-4718433 and one drop of the2-methylbutane emulsion from Example 1(j) were placed on a microscopeobject glass at 20ΕC and investigated at 400× magnification. Significantgrowth of microbubbles was observed as the drops mixed.

q) A sample of the perfluorobutane gas dispersion from Example 1(a) wasdiluted with purified water (1:1) and cooled to 0ΕC. A drop of theperfluorodecalin/perfluorobutane emulsion from Example 1(z) was added tothe diluted microbubble dispersion on a microscope object glassmaintained at 0ΕC by means of a heating/cooling stage and covered with acover glass, also at 0ΕC. The temperature of the object glass wasgradually increased to 40ΕC using the heating/cooling stage. Rapid andsubstantial microbubble growth was observed by microscopy.

r) A sample of the perfluorobutane gas dispersion from Example 1(a) wasdiluted with purified water (1:1) and cooled to 0ΕC. A drop of theperfluorodecalin/perfluoropropane emulsion from Example 1(aa) was addedto the diluted microbubble dispersion on a microscope object glassmaintained at 0ΕC by means of a heating/cooling stage and covered with acover glass, also at 0ΕC. The temperature of the object glass wasgradually increased to 40ΕC using the heating/cooling stage. Rapid andsubstantial microbubble growth was observed by microscopy.

s) A sample of the perfluorobutane gas dispersion from Example 1(a) wasdiluted with purified water (1:1) and cooled to 0ΕC. A drop of theperfluorodecalin/sulphur hexafluoride emulsion from Example 1(ab) wasadded to the diluted microbubble dispersion on a microscope object glassmaintained at 0ΕC by means of a heating/cooling stage and covered with acover glass, also at 0ΕC. The temperature of the object glass wasgradually increased to 40ΕC using the heating/cooling stage, whereuponan increase in the size of the microbubbles was observed after 4-5minutes, although the increase was less heavy and less rapid then thatobserved in Examples 2(q) and 2(r).

t) A sample of the perfluorobutane gas dispersion from Example 1(a) wasdiluted with purified water (1:1) and cooled to 0ΕC. A drop of thePluronic F68-stabilised perfluoropentane emulsion from Example 1(ad) wasadded to the diluted microbubble dispersion on a microscope object glassmaintained at 0ΕC by means of a heating/cooling stage and covered with acover glass, also at 0ΕC. The temperature of the object glass wasgradually increased to 40ΕC using the heating/cooling stage. Rapid andsubstantial microbubbble growth was observed by microscopy.

u) One drop of the perfluorobutane gas dispersion from Example 1(a) andone drop of the Brij58:Fluorad FC-17° C.-stabilised perfluoropentaneemulsion from Example 1(aj) were placed on a microscope object glasswarmed to 40° C. and investigated at 400× magnification. After a whileslow microbubble growth was observed.

v) One drop of the perfluorobutane gas dispersion from Example 1(a) andone drop of the Brij58:Fluorad FC-170C-stabilised perfluoropentaneemulsion from Example 1(ak) were placed on a microscope object glasswarmed to 40° C. and investigated at 400× magnification. After a whilemicrobubble growth was observed.

w) One drop of the perfluorobutane gas dispersion from Example 1(a) andone drop of the perfluoro-4-methylpent-2-ene emulsion from Example 1(al)were placed on a microscope object glass warmed to 40° C. andinvestigated at 400× magnification. After a while slow microbubblegrowth was observed.

x) One drop of the perfluorobutane gas dispersion from Example 1(a) andone drop of the 1H,1H,2H-heptafluoropent-1-ene emulsion from Example1(am) were placed on a microscope object glass warmed to 40° C. andinvestigated at 400× magnification. Significant and rapid microbubblegrowth was observed as the drops mixed.

y) One drop of the perfluorobutane gas dispersion from Example 1(a) andone drop of the perfluorocyclopentene emulsion from Example 1(an) wereplaced on a microscope object glass warmed to 40° C. and investigated at400× magnification. Significant, rapid and heavy microbubble growth wasobserved as the drops mixed.

z) 400 μl of a perfluorobutane gas dispersion prepared as in Example1(b) was transferred to a 2 ml vial at room temperature, and 100 μl ofthe azeotropic emulsion of Example 1(ap) was added. One droplet of themicrobubble/emulsion mixture was placed on a microscope object glassmaintained at 20ΕC by means of a heating/cooling stage. The temperatureof the object glass was rapidly raised to 37° C. using theheating/cooling stage. A substantial, spontaneous and rapid microbubblegrowth was observed.

aa) One drop of biotinylated microbubbles prepared as described inExample 1(bq) was added to one drop of emulsion prepared as described inExample 1(bh) on a microscope object glass warmed up to 60ΕC andinvestigated at 400× magnification. Significant growth of microbubblesand accumulation of microbubbles at the aggregated emulsion droplets wasseen.

ab) Microbubbles prepared as described in Example 1(bq) may be analysedby flow cytometry, e.g. by employing a fluorescent streptavidin emulsionprepared as described in Example 1(bk) to detect attachment ofstreptavidin to the biotinylated microbubbles.

ac) One drop of the Echovist dispersion prepared as described in Example1(bo) was placed on an object glass for microscopy investigation andkept at 37ΕC using a heating/cooling stage. The sample was covered witha cover glass and placed under a microscope. Significant bubble growthwas observed.

ad) One drop of the aerogel dispersion from Example 1(br) was placed onan object glass for microscopy investigation and kept at 37ΕC using aheating/cooling stage. The sample was covered with a cover glass andplaced under a microscope. A droplet of 2-methylbutane emulsion (fromExample 1(c) above, except that 100 μl 2-methylbutane was used insteadof 200 μl) was added to the edge of the cover glass so that the emulsionpenetrated into the aerogel dispersion. On increasing the temperature toapproximately 60ΕC, microbubbles occurred from the aerogel particles.

ae) [comparative] One drop of the aerogel dispersion from Example 1(br)was placed on an object glass for microscopy investigation and kept at20ΕC using a heating/cooling stage. The sample was covered with a coverglass and placed under a microscope and the temperature was raised to60ΕC. No microbubble growth was observed.

af) One drop of the microbubble dispersion from Example 1(bs) was placedon an object glass for microscopy investigation. The sample was coveredwith a cover glass and placed under a microscope fitted with aheating/cooling stage keeping the sample temperature at 20ΕC. Onedroplet of 2-methylbutane emulsion from Example 1(c) above was added tothe edge of the cover glass so that the emulsion penetrated themicrobubble dispersion. No microbubble growth was observed during themixing stage. The temperature was then raised to 40ΕC, whereuponsubstantial microbubble growth was observed.

ag) [comparative] One drop of the microbubble dispersion from Example1(bs) was placed on an object glass for microscopy investigation. Thesample was covered with a cover glass and placed under a microscopefitted with a heating/cooling stage keeping the sample temperature at20ΕC. When the temperature was raised to 40ΕC, no microbubble growth wasobserved.

ah) To Echovist granulate (Schering AG) on a microscope object glass wasadded one drop of solvent for Echovist granulate at ambient temperature.One drop of 2-methylbutane emulsion prepared as Example 1(bw) was addedand investigated at 100× magnification. Significant growth ofmicrobubbles was observed as the drops mixed.

ai) One drop of Levovist⁷ prepared for injection and one drop of2-methylbutane emulsion prepared as in Example 1(bw) were placed on amicroscope object glass at ambient temperature and investigated at 400×magnification. Significant, rapid and heavy growth of microbubbles wasobserved as the drops mixed.

aj) One drop of perfluorobutane gas dispersion from Example 1(br) andone drop of 2-methylbutane emulsion prepared as Example 1(bw) wereplaced on a microscope object glass at ambient temperature andinvestigated at 400× magnification. Significant, rapid and heavymicrobubble growth was observed as the drops mixed.

ak) One drop of 2-methylbutane emulsion prepared as Example 1(by) wasadded to one drop of Buckminsterfullerene C₆₀ dispersion from Example1(bu) on a microscope object glass at 40° C. Significant, heavy andrapid growth of microbubbles was observed as the drops mixed.

al) One drop of 2-methylbutane emulsion prepared as Example 1(bw) wasadded to one drop of sulphur hexafluoride gas dispersion from Example1(by) on a microscope object glass at 40° C. Significant, rapid andheavy microbubble growth was observed as the drops mixed.

am) One drop of 0.5M hydrochloric acid was added to one drop ofperfluorobutane gas dispersion in aqueous sodium bicarbonate fromExample 1(bx) on a microscope object glass at ambient temperature.Rapid, heavy and short lived microbubble growth was observed as thedrops mixed.

an) One drop of 2-methylbutane emulsion prepared as in Example 1(bw) wasadded to one drop of the perfluorobutane gas dispersion with iron oxideparticles from Example 1(bz) on a microscope object glass at ambienttemperature. Significant, heavy and rapid microbubble growth wasobserved as the drops mixed.

ao) One drop of 2-methylbutane emulsion prepared as in Example 1(bw) wasadded to one drop of the perfluorobutane gas dispersion with iron oxideparticles from Example 1(ca) on a microscope object glass at ambienttemperature. Significant, heavy and rapid microbubble growth wasobserved as the drops mixed.

ap) [comparative] One drop of 2-methylbutane emulsion prepared asExample 1(bw) was added to one drop of the iron oxide particledispersion from Example 1(cb) on a microscope object glass at ambienttemperature. No microbubble formation was observed.

aq) One drop of 2-methylbutane emulsion prepared as in Example 1(bw) wasadded to one drop of the perfluorobutane gas dispersion with oleicacid-coated iron oxide particles from Example 1(cc) on a microscopeobject glass at ambient temperature. Significant, heavy and rapidmicrobubble growth was observed as the drops mixed.

ar) 1 ml of the microbubble dispersion prepared as described in Example1(bp) was diluted with 50 ml water. One drop of the diluted dispersionwas added to one drop of soda water on a microscope object glass atambient temperature. Spontaneous microbubble growth was observed as thedrops mixed.

as) 0.4 μl of a biotinylated microbubble dispersion prepared accordingto Example 1(bq) and 0.02 ml of perfluorodimethylcyclobutane emulsionprepared as described in Example 1(bh) are added sequentially to abeaker containing 200 ml of Isoton at 37ΕC with continuous stirring. Themixture is incubated for 20 seconds. A beam of pulsed ultrasound (10 kHzrepetition frequency, 100 μJ in each pulse) at 2.5 MHz is aimed throughthe solution, which is obsreved in sharp side light against a blackbackground. A bright streak of larger bubbles is immediately observed inthe beam path.

at) One drop of the microbubble dispersion prepared as in Example 1(bl)is placed on an object glass for microscopy examination. The sample iscovered with a cover glass and placed under a microscope fitted with aheating/cooling stage, keeping the temperature at 20ΕC. One droplet of aperfluorodimethylcyclobutane emulsion prepared as in Example 1(as) isadded to the edge of the cover glass so that the emulsion can penetratethe microbubble dispersion. On increasing the temperature toapproximately 60ΕC, substantial microbubble growth is observed.

EXAMPLE 3 In Vitro Microbubble Size and Distribution Characterisation

A) Measurements Using Malvern Mastersizer

Microbubble growth and the change in size distribution following mixturewith diffusible component were analysed using a Malvern Mastersizer 1002fitted with a 45 mm lens and having a measuring range of 0.1-80 μm. Thesample cell contained Isoton II (150 ml) and was connected to athermostatted bath operable over the temperature range 9-37ΕC. A sampleof the perfluorobutane gas dispersion from Example 1(a) (110 μl) wasadded to the sample cell and after equilibration a portion of the2-methylbutane emulsion from Example 1(c) (500 μl) was added. The IsotonII solution was pumped through the Mastersizer and the thermostattedbath so as to pass the measuring cell every 30 seconds. Repeatedmeasurements were carried out every 30 seconds for 3 minutes. Thetemperature of the Isoton II solution was gradually increased andfurther measurements were made. The perfluorobutane gas dispersion andthe 2-methylbutane emulsion were also analysed separately using similarconditions.

Analysis of the perfluorobutane gas dispersion alone showed that at 9ΕC82% of the microbubbles were of size below 9.9 μm; this proportion wasreduced to 31% when the temperature had increased to 37ΕC. Thistemperature change was accompanied by a correspnding increase in theproportion of microbubbles in the size range 15-80 μm, from 8% to 42%.

Following mixing of the perfluorobutane gas dispersion and2-methylbutane emulsion at 9ΕC a slight increase in microbubble size wasobserved. Increase of the temperature to 25ΕC led to strong microbubblegrowth, with about 81% of the microbubbles having sizes in the range15-80 μm. Further temperature increase led to microbubble growth tosizes beyond the measuring range of the instrument.

Mixing of the perfluorobutane gas dispersion and 2-methylbutane emulsionat 37ΕC led to rapid microbubble growth; after one 30 second measuringcycle 97% of the microbubbles had sizes in the range 15-80 μm.

b) Measurements Using Coulter Multisizer

Microbubble growth and the change in size distribution following mixturewith diffusible component were analysed using a Coulter Multisizer IIfitted with a 50 μm aperture and having a measuring range of 1-30 μm.The two components of each sample were added to the sample cell, whichcontained 200 ml Isoton II preheated to 37ΕC, at which temperature themeasurements were performed. The size distribution of the mixture wasmeasured immediately and 1.5 minutes after introduction of the samples.Thereafter the sample cell was exposed to ultrasound for 1 minute, usinga 2.25 MHz transducer connected to a pulse generator; the energy levelwas 100 μJ.

b)(i) Mixing of the perfluorobutane gas dispersion from Example 1(ag)and perfluoropentane emulsion from Example 1(l) led to rapid andsubstantial microbubble growth after exposure to ultrasound. The totalvolume concentration increased from 3% to approximately 16%.

b)(ii) Mixing of the perfluorobutane gas dispersion from Example 1(ag)and perfluorobutane emulsion from Example 1(m) led to rapid andsubstantial microbubble growth. The total volume concentration increasedfrom 1% to approximately 6%.

b)(iii) Mixing of the perfluorobutane gas dispersion from Example 1(ag)and perfluoropentane emulsion from Example 1(p) led to rapid andsubstantial microbubble growth after exposure to ultrasound. The totalvolume concentration increased from approximately 1% to approximately4%.

b)(iv) Mixing of the perfluorobutane gas dispersion from Example 1(ag)and perfluoropentane emulsion from Example 1(af) led to rapid andsubstantial microbubble growth after exposure to ultrasound. The totalvolume concentration increased from approximately 2% to approximately8%.

b)(v) Mixing of the perfluorobutane gas dispersion from Example 1(ag)and perfluoropentane/perfluoro-4-methylpent-2-ene emulsion from Example1(q) led to rapid and substantial microbubble growth after exposure toultrasound. The total volume concentration increased from 2% toapproximately 4%.

b)(vi) Mixing of the perfluorobutane gas dispersion from Example 1(ag)and perfluoropentane/1H,1H,2H-heptafluoropent-1-ene emulsion fromExample 1(r) led to rapid and substantial microbubble growth. The totalvolume concentration increased from 2% to approximately 4.5%.

b)(vii) Mixing of the perfluorobutane gas dispersion from Example 1(ag)and perfluoropentane emulsion from Example 1(s) led to rapid andsubstantial microbubble growth after exposure to ultrasound. The totalvolume concentration increased from 2% to approximately 13%.

b)(viii) Mixing of the perfluorobutane gas dispersion from Example 1(ag)and perfluoropentane emulsion from Example 1(t) led to rapid andsubstantial microbubble growth after exposure to ultrasound. The totalvolume concentration increased from 2% to approximately 13%.

b)(ix) Mixing of the perfluorobutane gas dispersion from Example 1(ag)and perfluoropentane emulsion from Example 1(u) led to rapid andsubstantial microbubble growth after exposure to ultrasound. The totalvolume concentration increased from 3% to approximately 15%.

b)(x) Mixing of the perfluorobutane gas dispersion from Example 1(ag)and perfluoropentane emulsion from Example 1(v) led to rapid andsubstantial microbubble growth after exposure to ultrasound. The totalvolume concentration increased from 3% to approximately 22%.

b)(xi) Mixing of the perfluorobutane gas dispersion from Example 1(ag)and perfluoropentane emulsion from Example 1(ai) led to rapid andsubstantial microbubble growth after exposure to ultrasound. The totalvolume concentration increased from approximately 3% to approximately8%.

b)(xii) Mixing of the perfluorobutane gas dispersion from Example 1(ag)and perfluoropentane:perfluoro-4-methylpent-2-ene emulsion from Example1(x) led to rapid and substantial microbubble growth after exposure toultrasound. The total volume concentration increased from 2% toapproximately 7.5%.

b)(xiii) Mixing of the perfluorobutane gas dispersion from Example 1(ag)and perfluoropentane:perfluoro-4-methylpent-2-ene emulsion from Example1(y) led to rapid and substantial microbubble growth after exposure toultrasound. The total volume concentration increased from 2.5% toapproximately 7%.

b)(xiv) Mixing of the perfluorobutane gas dispersion from Example 1(ag)and perfluoropentane emulsion from Example 1(ac) led to rapid andsubstantial microbubble growth. The increase in the size of themicrobubbles was more heavy and more rapid then that observed in Example3(b)(xv). The total volume concentration increased from 3.5% toapproximately 53%.

b)(xv) Mixing of the perfluorobutane gas dispersion from Example 1(ag)and perfluoropentane emulsion from Example 1(ae) led to rapid andsubstantial microbubble growth. The total volume concentration increasedfrom 7% to approximately 19%. Exposure to ultrasound resulted in furthermicrobubble growth indicated by an increase in the total volumeconcentration to approximately 54.5%.

b)(xvi) Mixing of the perfluoropropane gas dispersion from Example 1(ah)and perfluoropentane emulsion from Example 1(l) led to rapid microbubblegrowth, although not so heavy as observed in Example 3(b)(i). The totalvolume concentration increased from 3% to approximately 4.5%.

b)(xvii) Mixing of the perfluorobutane gas dispersion from Example 1(ag)and perfluoropentane emulsion from Example 1(o) led to rapid andsubstantial microbubble growth after exposure to ultrasound. The totalvolume concentration increased from approximately 1% to approximately8%.

b)(xviii) A sample of perfluorohexane emulsion prepared as described inExample 1(ar) had a total concentration of droplets of 8.6 vol % and thedroplet size was 2.6 μm.

b)(xix) A sample of 2,2,3,3,3-pentafluoropropyl methyl ether emulsionprepared as described in example 1(at) had a total concentration ofdroplets of 4.3 vol % and the droplet size was 1.5 μm.

b)(xx) A sample of 2H,3H-decafluoropentane emulsion prepared asdescribed in Example 1(au) had a total concentration of droplets of 5.6vol % and the droplet size was 1.9 μm.

b)(xxi) A sample of perfluoroheptane emulsion prepared as described inExample 1(bj) had a total concentration of droplets of 8.5 vol % and thedroplet size was 2.2 μm.

(c) Measurements Using Coulter Multisizer (140 μm Aperture)

Microbubble growth and change of size distribution following mixturewith diffusible component emulsions were analysed using a CoulterMultisizer II fitted with a 140 μm aperture. The measuring range was setto 10-80 μm. The bubble dispersion and emulsion droplets were added tothe sample cell containing 200 ml preheated Isoton II. The measurementswere performed at 37ΕC. The size distribution of the mixture wasmeasured immediately and 3 minutes after mixing. Thereafter the samplesolution was exposed to ultrasound for 1 minute using a 2.25 MHztransducer connected to a pulse generator. The energy level was 100 μJ.The size distribution of the mixture was measured 1 minute and 3 minutesafter exposure to ultrasound.

c)(i) Following addition of 182 μl of the heptafluoropent-1-ene emulsionprepared as described in Example 1(am) to 400 μl of a perfluorobutanegas dispersion prepared as described in Example 1(bl), the microbubblesimmediately increased in size and the total volume concentration in thesize range 10-80 μm was increased from insignificant to about 60 vol %within 1 minute.

c)(ii) Following addition of 70 μl of the perfluorodimethylcyclobutaneemulsion prepared as described in Example 1(av) to 330 μl ofperfluorobutane gas dispersion prepared as described in Example 1(bl),the microbubbles increased substantially in size after exposure toultrasound. The total volume concentration in the size range 10-80 μmwas increased from insignificant to about 14 vol % within 3 minutes.

c)(iii) Following addition of 71 μl of the perfluorodimethylcyclobutaneemulsion prepared as described in Example 1(aw) to 330 μl ofperfluorobutane gas dispersion prepared as described in Example 1(bl),the microbubbles increased substantially in size after exposure toultrasound. The total volume concentration in the size range 10-80 μmwas increased from insignificant to about 8.6 vol % within 3 minutes.

c)(iv) Following addition of 105 μl of the perfluorodimethylcyclobutaneemulsion prepared as described in Example 1(ax) to 300 μl ofperfluorobutane gas dispersion prepared as described in Example 1(bl),the microbubbles increased in size after exposure to ultrasound. Thetotal volume concentration in the size range 10-80 μm was increased from3.2 vol % to about 4.8 vol % within 3 minutes.

c) (v) Following addition of 105 μl of the perfluorodimethylcyclobutaneemulsion prepared as described in Example 1(ay) to 300 μl ofperfluorobutane gas dispersion prepared as described in Example 1(bl),the microbubbles increased in size after exposure to ultrasound. Thetotal volume concentration in the size range 10-80 μm was increased from1.5 vol % to about 2.2 vol % within 3 minutes.

c) (vi) Following redispersion of lyophilised perfluorobutanemicrobubbles in perfluorodimethyl-cyclobutane emulsion as described inExample 1(bg) an immediate increase in microbubble size occurred. Thetotal volume concentration in the size range 10-80 μm was increased frominsignificant to about 60 vol % within 1 minute.

c)(vii) Following addition of 76 μl of the 1H-tridecafluorohexaneemulsion prepared as described in Example 1(bi) to 400 μl of aperfluorobutane gas dispersion prepared as described in Example 1(bl),the microbubbles immediately increased in size and the total volumeconcentration in the size range 10-80 μm was increased frominsignificant to about 20 vol % within 3 minures.

c)(viii) Following addition of 63 μl of the perfluorodimethylcyclobutaneemulsion prepared as described in Example 1(bm) to 741 μl of aperfluorobutane gas dispersion prepared as described in Example 1(bl)the microbubbles immediately increased in size and the total volumeconcentration in the size range 10-80 μm was increased frominsignificant to about 2 vol % within 3 minutes.

c)(ix) Following addition of 67 μl of the perfluorodimethylcyclobutaneemulsion prepared as described in Example 1(aq) to 56 μl ofperfluoropropane gas dispersion prepared as described in Example 1(bn)the microbubbles increased in size after exposure to ultrasound. Thetotal volume concentration in the size range 10-80 μm was increased frominsignificant to about 2.7 vol % within 1 minute.

EXAMPLE 4 In Vitro Measurements of Acoustic Attenuation

a) A sample of the perfluorobutane gas dispersion from Example 1(a) (1μl) was suspended in Isoton II (55 ml) at 37ΕC and acoustic attenuationwas measured as a function of time using two broadband transducers withcentre-frequencies of 3.5 MHz and 5.0 MHz in a pulse-echo technique.After 20 seconds a diffusible component was added to the suspension andmeasurements were continued for a further 120 seconds.

a)(i) Following addition of 100 μl of the 2-methylbutane emulsion fromExample 1(c) attenuation immediately increased by a factor of more than4; exact quantification was not possible since the attenuation exceededthe maximum value measurable by the system. The effect lasted for 50seconds and was accompanied by a complete change in the shape of theattenuation spectra indicating a pronounced increase in microbubblesize.

a)(ii) Addition of 20 μl of the 2-methylbutane emulsion from Example1(c) led to a gradual increase in attenuation, reaching a maximum ofbetween three and four times the initial value after 40 seconds and thendecreasing rapidly. Again a complete change in the shape of theattenuation spectra indicated a pronounced increase in microbubble size.

a)(iii) Addition of 5 μl of the 2-methylbutane emulsion for Example 1(c)led to a gradual increase in attenuation, reaching a maximum of about50% above the initial value after 30 seconds and then decreasing slowlytowards the initial value. A shift towards lower resonance frequenciesin the attenuation spectra indicated a moderate increase in microbubblesize.

a)(iv) Addition of 500 μl of the 2-chloro-1,1,2-trifluoroethyldifluoromethyl ether emulsion from Example 1(e) led to a gradualincrease in attenuation, reaching a maximum of about 50% above theinitial value after 20 seconds and then decreasing slowly towards theinitial value. A shift towards lower resonance frequencies in theattenuation spectra indicated a moderate increase in microbubble size.

a)(v) Addition of 500 μl of the perfluoropentane emulsion from Example1(d) led to a small increase in attenuation. A shift towards lowerresonance frequencies in the attenuation spectra indicated a smallincrease in microbubble size.

By way of control, addition of 500 μl of water produced no discerniblechange in attenuation.

b) A sample of the 2-methylbutane emulsion from Example 1(c) (100 μl)was added to the Isoton II (55 ml) at 37ΕC and acoustic attenuation wasmeasured as described in (a) above. After 20 seconds a sample of theperfluorobutane gas dispersion from Example 1(a) (1 μl) was added to thesuspension and measurements were continued for a further 120 seconds.Attenuation increased rapidly following addition of the gas dispersion,reaching the maximum measuring level of the system after 20 seconds, andstarting to decrease after 50 seconds. The attenuation spectra indicatedthe presence of large microbubbles.

By way of control, when 100 μl of water was used in place of the2-methylbutane emulsion, attenuation increased rapidly followingaddition of the gas dispersion; after 40 seconds it reached a stablelevel one quarter of that measured using the 2-methylbutane emulsion.Attenuation remained at this level throughout the remainder of the 120second measurement period. The attenuation spectra indicated thepresence of small microbubbles.

EXAMPLE 5 In Vivo Imaging of Dog Heart with Perfluorobutane GasDispersion [Comparative]

An injection syringe containing an amount of the perfluorobutane gasdispersion from Example 1(b) corresponding to 2 μl of gas content wasprepared and the contents were injected into an open-chest 20 kg dogusing a catheter inserted into an upper limb vein. Imaging of the heartwas performed with a Vingmed CFM-750 scanner, using a midline short axisprojection. The scanner was adjusted to acquire images once in eachend-systole by gating to the ECG of the animal. Bright contrast was seenin the right ventricle a few seconds after the injection, and contrastof similar brightness appeared in the left ventricle some 4-5 secondslater, however with a substantial attenuation transiently hiding theposterior parts of the heart. Off-line digital backscatter intensityanalysis was performed based on cine-loop data recorded by the scanner.A brief, transient peak of contrast enhancement lasting some 10 seconds,beginning 3 seconds after the onset of contrast enhancement within theleft ventricle was evident in a representative region of anterior leftventricle myocardium.

EXAMPLE 6 In Vivo Imaging of Dog Heart with 2-methylbutane Emulsion[Comparative]

An injection syringe containing 1.0 ml of the 2-methylbutane emulsionfrom Example 1(c) was prepared and the contents were injected into theanimal as in Example 5. Imaging of the heart was performed as describedin Example 5. No contrast effects could be seen.

EXAMPLE 7 In Vivo Imaging of Dog Heart with Perfluorobutane GasDispersion and 2-methylbutane Emulsion

Injection syringes were prepared as in Examples 5 and 6 and the contentsof both syringes were injected simultaneously into the dog via a Y-piececonnector and the catheter described in Example 5. Imaging of the heartwas performed as described in Example 5. The echo enhancement of theventricles was similar to the observations in Example 5. In the leftventricular myocardium there was a monotonous rise in echo intensity inthe 30 seconds following arrival of the contrast bolus to the coronarycirculation. The contrast effects in the myocardium had completelyvanished 5 minutes later.

EXAMPLE 8 In Vivo Imaging of Dog Heart with Perfluoropentane Emulsion[Comparative]

An injection syringe containing 0.5 ml of the perfluoropentane emulsionfrom Example 1(d) was prepared and the contents were injected into theanimal as in Example 5. Imaging of the heart was performed as describedin Example 5. No signs of echo enhancement could be observed in anyregion of the image.

EXAMPLE 9 Low Intensity In Vivo Imaging of Dog Heart withPerfluorobutane Gas Dispersion and Perfluoropentane Emulsion

Injection syringes were prepared as in Examples 5 and 8 and the contentsof both syringes were injected simultaneously into an open-chest 20 kgmongrel dog via a Y-piece connector and a catheter inserted into anupper limb vein.

Imaging of the heart was performed with a Vingmed CFM-750 sanner, usinga midline short axis projection. The scanner was adjusted to minimiseacoustical output by lowering the emitted power to a value of 1 (on ascale ranging from 0 to 7), and by acquiring images only once in eachend-systole by gating to the ECG of the animal. The observed contrastenhancement was as described in Example 5 with, however, a slightlyprolonged duration in the myocardium.

EXAMPLE 10 High Intensity In Vivo Imaging of Dog Heart withPerfluorobutane Gas Dispersion and Perfluoropentane Emulsion

The experiment of Example 9 was repeated, except that the scanner outputwas adjusted to maximise ultrasound exposure to the imaged tissueregion. This was done by using a combination of continuous high framerate imaging and the highest output power (7 on a scale ranging from 0to 7). After the injection, intense and bright contrast enhancement wasseen in both ventricles of the heart. A steady rise in contrastenhancement was seen in all regions of the myocardium, up to anenhancement intensity approaching the maximum white level on the screen.The duration of tissue contrast was approximately 30 minutes, whilstcontrast effects in the blood-pool declined to near baseline within 5minutes of the injection, leaving an image with almost no blood-poolattenuation, and a complete and extremely bright circumferentialcontrast enhancement of the myocardium. The contrast effect in themyocardium close to the transducer did not seem to fade despitecontinuous high intensity ultrasound exposure.

EXAMPLE 11 High Intensity In Vivo Imaging of Dog Heart withPerfluorobutane Gas Dispersion and Perfluoropentane Emulsion

The procedure of Example 10 was repeated except that theperfluoropentane emulsion employed was prepared by cooling a solution ofpolyethylene glycol 10000 methyl ether 16-hexadecanoyloxyhexadecanoate(200 mg, prepared as in Example 2(k) of WO-A-9607434) in purified water(20 ml), transferring a 1 ml portion of this solution to a 2 ml vial,adding perfluoropentane (200 μl), shaking the vial for 45 seconds usinga CapMix7 and storing the emulsion at 0ΕC when not in use. The observedcontrast enhancements of blood and myocardial tissue were as describedin Example 5.

EXAMPLE 12 In Vivo Imaging of Dog Kidney

The same substances and injection procedure as described in Example 9were used. The left kidney of the dog was imaged through the intactabdominal wall using the same high output instrument settings as inExample 10. Central structures of the kidney containing the supplyingarteries were included in the image. 20 seconds after the injection, thebeginning of a steady rise in kidney parenchymal contrast enhancementwas seen, reaching an intensity plateau of extreme brightness 1-2minutes later. The transducer was moved to image the right kidney 4minutes after the injection. At first, this kidney had a normal,non-enhanced appearance. However, this application of high intensityultrasound was observed to generate a slight increase in echo intensityafter a few minutes, although not up to the level that was observed inthe left kidney.

EXAMPLE 13 In Vivo Imaging of Dog Heart with Perfluorobutane GasDispersion and Reduced Amount of Perfluoropentane Emulsion

The procedure of Example 10 was repeated except that the dose of theperfluoropentane emulsion was reduced to one third. The peak intensityof myocardial contrast enhancement was comparable to that observed inExample 10, but the duration of tissue contrast was reduced from 30minutes to less than 10 minutes.

EXAMPLE 14 Closed-Chest In Vivo Imaging of Dog Heart withPerfluorobutane Gas Dispersion and Perfluoropentane Emulsion

The procedure of Example 10 was repeated in a closed-chest experiment.The myocardial contrast enhancement was comparable to that observed inExample 10.

EXAMPLE 15 Colour Doppler In Vivo Imaging of Dog Heart withPerfluorobutane Gas Dispersion and Perfluoropentane Emulsion

The procedure of Example 10 was repeated except that the scanner (in thecolour Doppler mode) was applied to the left heart ventricle during thefirst minute after injection in order to initiate microbubble growth.Thereafter the myocardial contrast enhancement was more intense thanthat observed in Example 10.

EXAMPLE 16 In Vivo Imaging of Dog Heart with Perfluorobutane GasDispersion and perfluoro-4-methylpent-2-ene Emulsion

0.5 ml of isotonically reconstituted perfluorobutane gas dispersionprepared in accordance with Example 1(ag) and 66 μl of theperfluoro-4-methylpent-2-ene emulsion from Example 1(al) were injectedas described in Example 10. The resulting myocardial contrastenhancement was comparable in intensity to that observed in Example 10,but had a duration of 6-8 minutes.

EXAMPLE 17 In Vivo Imaging of Hyperemic Region of Dog Heart withPerfluorobutane Gas Dispersion and Perfluoropentane Emulsion

A branch of the circumflex coronary artery of the dog was temporarilyligated for 2 minutes, whereafter contrast agent was injected asdescribed in Example 10. Contrast enhancement of the now hyperaemicmyocardium was substantially more intense than that of surroundingnormal tissue.

EXAMPLE 18 In Vivo Imaging of Dog Heart with Perfluorobutane GasDispersion and Perfluorodimethyl-Cyclobutane Emulsion

0.5 ml of isotonically reconstituted perfluorobutane gas dispersionprepared in accordance with Example 1(ag) and 66 μl of theperfluorodimethylcyclobutane emulsion from Example 1(ao) were injectedas described in Example 10. The resulting intense myocardial contrastenhancement was comparable to that observed in Example 16.

EXAMPLE 19 In Vivo Imaging of Dog Heart with Perfluorobutane GasDispersion and Perfluorodimethyl-Cyclobutane Emulsion

0.5 ml of isotonically reconstituted perfluorobutane gas dispersionprepared in accordance with Example 1(bl) and 66 μl of theperfluorodimethylcyclobutane emulsion from Example 1(aq) were injectedas described in Example 10. The resulting intense myocardial contrastenhancement was comparable to that observed in Example 16.

EXAMPLE 20 In Vivo Aparticle-To-Particle≅ Targeting

0.02 μl/kg of avidinylated perfluorobutane microbubbles preparedaccording to Example 1(bq), and 0.02 μl/kg ofperfluorodimethylcyclobutane emulsion prepared as described in Example1(bh) are simultaneously intravenously injected into a 20 kganaesthetised mongrel dog, while the heart is imaged by ultrasound asdescribed in Example 10.

Myocardial echo enhencement was similar to that observed in Example 10,except that the peak of attenuation in the left ventriclar blood was farless pronounced.

EXAMPLE 21 In Vivo Imaging of Rabbit Heart with Perfluorobutane GasDispersion and Perfluorodimethylcyclobutane Emulsion

An injection syringe containing an amount of the perfluorobutanemicrobubble dispersion prepared as in Example 1(bl) (volume mediandiameter 3.0 μm) corresponding to 1 μl of gas content and a furtherinjection syringe containing 105 μl of the perfluorodimethylcyclobutaneemulsion from Example 1(aq) were prepared. The contents of both syringeswere injected simultaneously into a 5 kg rabbit using a catheterinserted into an ear vein. B-mode imaging of the heart was performedusing an ATL HDI-3000 scanner with a P5-3 probe, using an open thoraxparasternal short axis projection. The results were comparable to thoseobserved in Example 18.

EXAMPLE 22 Ultrasonication-Induced Drug Delivery

A 3 kg anaesthetised New Zealand Black rabbit was injected intravenouslywith 0.04 ml of perfluorodimethylcyclobutane emulsion prepared asdescribed in Example 1(aq) and simultaneously with 0.12 ml ofperfluorobutane gas suspension prepared as described in Example 1(bl),while the left kidney was imaged with an ATL HDI-3000 scanner with aP5-3 probe, the scanner being adjusted for maximum output power.Significant bubble growth and accumulation within the kidney parenchymawas observed. Then 160 mg of FITC-dextran (mw 2,000,000) was dissolvedin 5 ml of water and injected intravenously, and ultrasound imaging atthe same site was continued for another 5 minutes, now swithcing thescanner to Power Doppler mode to maximise acoustical output. The animalwas then sacrificed, and both kidneys were removed and examined inultraviolet light. An increased amount of fluorescence was observed as50-100 μm spots in the interstitium within the regions of the leftkidney that were exposed to imaging ultrasound in the presence ofmicrobubbles. Associated with each such spot was a nephron devoid ofintravascular fluorescence.

EXAMPLE 23 Albunex7 as Gas Dispersion

0.3 ml/kg of Albunex7 and 1.5 μl/kg of perfluorodimethylcyclobutaneemulsion prepared as described in Example 1(aq) were injectedintravenously into a 20 kg anaesthetised male mongrel dog and imaged byultrasound as described in Example 10. The myocardial enhancement was asdescribed in Example 10.

EXAMPLE 24 Targeted Microbubbles in Imaging of Rabbit Heart

0.1 μl/kg of microbubbles prepared as described in Example 1(az) wereinjected intravenously into a rabbit, while imaging the rabbit's heartby ultrasound using an ATL HDI-3000 scanner with a P5-3 probe. A faintbut lasting myocardial echo enhancement was seen. Three minutes later,1.5 μl/kg of perfluorodimethylcyclobutane emulsion prepared as describedin Example 1(aq) was injected. A slight increase in the echo intensityfrom the insonified myocardium was observed.

EXAMPLE 25 In Vivo Imaging of Rat Heart with Perfluorobutane GasDispersion and Perfluorodimethylcyclobutane Emulsion

The experiment described in Example 19 was performed on a rat, withcomparable results.

EXAMPLE 26 In Vivo Imaging of Dog Heart with Perfluorobutane GasDispersion and Perfluorohexane Emulsion

0.1 μl/kg of perfluorohexane emulsion prepared as described in Example1(ar) and 0.2 μl/kg of the perfluorobutane microbubble suspensionprepared as decribed in Example 1(bl) were injected simultaneously intoa dog as described in Example 0.10. The myocardial contrast effect wascomparable to that observed in Example 10.

EXAMPLE 27 In Vivo Imaging of Dog Heart with Perfluorobutane GasDispersion and heptafluoropent-1-ene Emulsion

0.3 μl/kg of the perfluorobutane microbubble suspension prepared asdescribed in Example 1(bl) and 0.15 ml of the heptafluoropent-1-eneemulsion described in Example 1(am) were injected simultaneously into adog as described in Example 10. A relatively weak myocardial contrasteffect was observed, which was however more intense and morelong-lasting than that which was observed in Example 5.

EXAMPLE 28 In Vivo Imaging of Dog Heart with Perfluorobutane GasDispersion and Perfluorodimethylcyclobutane Emulsion Stabilised withSterilised Phospholipid

0.3 μl/kg of a perfluorobutane microbubble suspension prepared asdescribed in Example 1(bl) and 0.3 μl/kg of theperfluorodimethylcyclobutane emulsion prepared as described in Example1(bm) were injected simultaneously into a dog as described in Example19. A myocardial contrast effect comparable to that described in Example19 was observed.

EXAMPLE 29 In Vivo Imaging of Dog Heart with Perfluoropropane GasDispersion and Perfluorodimethylcyclobutane Emulsion

0.17 ml of the perfluoropropane microbubble suspension prepared asdescribed in Example 1(bn) and 0.3 μl/kg of theperfluorodimethylcyclobutane emulsion prepared as described in Example1(aq) were injected simultaneously into a dog as described in Example19. A myocardial contrast effect comparable to that described in Example19 was observed.

EXAMPLE 30 In Vivo Imaging of Dog Gastrointestinal Tract withPerfluorobutane Gas Dispersion and Perfluorodimethylcyclobutane Emulsion

20 ml of an emulsion of perfluorodimethylcyclobutane, prepared asdescribed in Example 1(aq) is given via a gastric tube to ananaesthetised dog. Thereafter a small amount (dose range 0.1-0.2 μlgas/kg) of a perfluorobutane microbubble dispersion prepared as inExample 1(a) is injected intravenously. An ultrasound imaging transduceris applied onto the abdominal wall, and localised microbubble growth inthe gastric wall capillary system provides enhanced contrast withimproved delineation of the mucosal contours.

EXAMPLE 31 In Vivo Imaging of Dog Gastrointestinal Tract withPerfluorobutane Gas Dispersion and Perfluorodimethylcyclobutane Emulsion

A perfluorobutane microbubble dispersion prepared as in Example 1(a) isgiven via a gastric tube to an anaesthetised dog. The dispersion isallowed to distribute evenly inside the gastric ventricle, as verifiedby ultrasound imaging. A small amount of an emulsion ofperfluorodimethylcyclobutane, prepared as described in Example1(aq)(dose range 0.2-1 μl perfluorocarbon/kg), is injectedintravenously. The ultrasound transducer is maintained on the region ofinterest; microbubble growth in the gastric fluid layers proximal to themucosal surfaces provides enhanced contrast with improved delineation ofthe mucosal contours.

EXAMPLE 32 In Vivo Imaging of Dog Heart with Perfluorobutane GasDispersion and Perfluorodimethylcyclobutane Emulsion and CoadministeredAdenosine

An occluding snare was placed around a major branch of the left anteriordescending coronary artery of an open-chest 22 kg dog and an ultrasoundtransit time flowmeter was placed immediately downstream of theoccluder, which was then adjusted to produce a steady 25% flow reductionfrom about 14 to 10 ml/min. The contents of three syringes, respectivelycontaining (i) an amount of a perfluorobutane microbubble dispersionprepared as in Example 1(bl) corresponding to 4.4 μl of gas content,(ii) an amount of the perfluorodimethylcyclobutane emulsion from Example1(aq) corresponding to 33 μl of the dispersedperfluorodimethylcyclobutane phase, and (iii) 3.0 mg adenosine dissolvedin 0.9% saline, were then intravenously injected as a simultaneousbolus; commencing 10 seconds later a further 3.0 mg of adenosinedissolved in 0.9% saline was injected slowly over 20 seconds. Imaging ofthe left ventricle of the heart was performed using an ATL HDI-3000scanner with a P5-3 probe; continuous ultrasonication at maximum powerwas applied for 1 minute to induce microbubble growth, whereafter themyocardium was examined using B-mode imaging. A clearly evidentdifference in gray scale levels could be seen between stenotic areas(brighter than baseline recordings) and normal areas (very much brighterthan baseline recordings).

1. A combined preparation for simultaneous, separate or sequential useas a contrast agent in ultrasound imaging, said preparation comprising:i) an injectable aqueous medium having gas dispersed therein; and ii) acomposition comprising a diffusible component capable of diffusion invivo into said dispersed gas so as at least transiently to increase thesize thereof.
 2. A combined preparation as claimed in claim 1 whereinthe dispersed gas comprises air, nitrogen, oxygen, carbon dioxide,hydrogen, an inert gas, a sulphur fluoride, selenium hexafluoride, anoptionally halogenated silane, a low molecular weight hydrocarbon, aketone, an ester, a halogenated low molecular weight hydrocarbon or amixture of any of the foregoing.
 3. A combined preparation as claimed inclaim 2 wherein the gas comprises a perfluorinated ketone,perfluorinated ether or perfluorocarbon.
 4. A combined preparation asclaimed in claim 3 wherein the perfluorocarbon comprises aperfluoroalkane, perfluoroalkene or perfluorocycloalkane.
 5. A combinedpreparation as claimed in claim 2 wherein the gas comprises sulphurhexafluoride or a perfluoropropane, perfluorobutane or perfluoropentane.6. A combined preparation as claimed in claim 1 wherein the dispersedgas is stabilised by a coalescence-resistant surface membrane, afilmogenic protein, a polymer material, a non-polymeric andnon-polymerisable wall-forming material or a surfactant.
 7. A combinedpreparation as claimed in claim 6 wherein said surfactant comprises atleast one phospholipid.
 8. A combined preparation as claimed in claim 7wherein at least 75% of the said surfactant material comprisesphospholipid molecules individually bearing net overall charge.
 9. Acombined preparation as claimed in claim 8 wherein at least 75% of thefilm-forming surfactant material comprises one or more phospholipidsselected from phosphatidylserines, phosphatidylglycerols,phosphatidylinositols, phosphatidic acids and cardiolipins. 10.(canceled)
 11. A combined preparation as claimed in claim 1 wherein thecomposition comprising the diffusible component is formulated foradministration cutaneously, subcutaneously, intramuscularly,intravenously or by inhalation.
 12. A combined preparation as claimed inclaim 1 wherein the composition comprising the diffusible componentfurther comprises a carrier liquid.
 13. A combined preparation asclaimed in claim 12 wherein the diffusible component is dispersed in anaqueous carrier liquid in the form of an oil-in-water emulsion ormicroemulsion.
 14. A combined preparation as claimed in claim 13 whereinthe diffusible component comprises an aliphatic ether, polycyclic oil,polycyclic alcohol, heterocyclic compound, aliphatic hydrocarbon,cycloaliphatic hydrocarbon or halogenated low molecular weighthydrocarbon.
 15. A combined preparation as claimed in claim 14 whereinthe diffusible component comprises a perfluorocarbon.
 16. A combinedpreparation as claimed in claim 15 wherein the perfluorocarbon comprisesa perfluoroalkane, perfluoroalkene, perfluorocycloalkane,perfluorocycloalkene or perfluorinated alcohol.
 17. A combinedpreparation as claimed in claim 16 wherein the diffusible componentcomprises perfluoropentane, perfluorohexane orperfluorodimethylcyclobutane.
 18. A combined preparation as claimed inclaim 13 wherein the emulsion is stabilised by a phospholipidsurfactant.
 19. A combined preparation as claimed in claim 18 wherein atleast 75% of the said phospholipid surfactant comprises moleculesindividually bearing net overall charge.
 20. A combined preparation asclaimed in claim 19 wherein at least 75% of the phospholipid surfactantis selected from phosphatidylserines, phosphatidylglycerols,phosphatidylinositols, phosphatidic acids and cardiolipins. 21.(canceled)
 22. A combined preparation as claimed in claim 1 whichfurther includes a vasodilator drug.
 23. A combined preparation asclaimed in claim 22 wherein said vasodilator drug is adenosine. 24.(canceled)
 25. (canceled)
 26. A method of generating enhanced images ofa human or non-human animal subject which comprises the steps of: i)injecting a physiologically acceptable aqueous medium having gasdispersed therein into the vascular system of said subject; ii) before,during or after injection of said aqueous medium administering to saidsubject a composition comprising a diffusible component capable ofdiffusion in vivo into said dispersed gas so as at least transiently toincrease the size thereof; and iii) generating an ultrasound image of atleast a part of said subject.
 27. (canceled)
 28. A method as claimed inclaim 26 wherein a vasodilator drug is coadministered to the subject.29. A method as claimed in claim 28 wherein said vasodilator drug isadenosine.